Compositions and methods for diagnosing and/or treating kidney injury

ABSTRACT

One aspect of the technology relates to methods, assays and kits to identify ischemia and ischemic injury, including kidney injury, and are useful in determining efficacy of cancer treatments. In particular, differential phosphorylation of the nucleophosmin (NPM) polypeptide is an early marker of ischemic injuries such as kidney injury, AKI and ischemic renal cell injury. Another aspect of the technology relates to compositions and methods for the treatment of ischemia and kidney injury, including NPM inhibitory agents, including, but not limited to NPM inhibitory peptides for the treatment of ischemia and kidney injury.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.16/265,413 filed Feb. 1, 2019, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application 62/625,536 filed Feb. 2, 2018 andProvisional Application 62/777,514, filed on Dec. 10, 2018, the contentsof each are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract No. NIHDK-053387 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

SEQUENCE LISTING

This instant application contains a Sequence Listing which has beenfiled electronically in ASCII format. Said ASCII copy, created Aug. 25,2021, is named 701586-091560USPT_SL.txt and is 10,084 byes in size.

TECHNICAL FIELD

The present disclosure described herein relates to compositions andmethods for the treatment of ischemia and kidney injury. In particular,differential phosphorylation of the nucleophosmin (NPM) polypeptide isan early marker of ischemic injuries such as kidney injury, AKI andischemic renal cell injury. Nucleophosmin inhibitory peptides can beused for the treatment of such ischemia and kidney injury.

BACKGROUND OF THE INVENTION

Ischemia/reperfusion injury is a leading cause of human AKI.1-3 AKIoccurs in 7%-10% of hospitalized patients at average risk for renalinjury4 and between 23% and 74% of high-risk patients.5,6 Even a modestincrease in serum creatinine of >0.5 mg/dl increases the length of stayand hospital cost as well as morbidity and mortality.4 In fact,mortality has been reported to be as high as 80% when the serumcreatinine acutely rises by >2 mg/dl.4 In children and adults, a singleAKI episode risks progressive kidney injury resulting in ESRD.7,8 Lessthan 60% of children survive >3-5 years after a single AKI episode.9Surviving children have a 60% chance of developing high BP, proteinuria,and/or CKD that ultimately requires dialysis or kidney transplantation.9

This is partly due to the facts that diagnostic tests of AKI and organdysfunction are insensitive and that ischemic renal injury lackseffective treatment.10 Tissue ischemia/reperfusion causes proximaltubule epithelial cell (PTEC) injury, a major contributor to organfailure.1,3,11 Experimental evidence implicates Bax, a quintessentialBCL2 proapoptotic protein, as an important cause of regulated PTEC deathduring hypoxic or ischemic stress and contributor to AKE11-15

As such, ischemic AKI lacks a urinary marker for early diagnosis and aneffective therapy. Accordingly, there is a need for more effective andaccurate biomarkers for kidney injury and AKI, as well as effectivetherapeutics for the treatment of kidney injury.

BRIEF DESCRIPTION OF THE INVENTION

The technology described herein relates to methods, compositions andkits for the diagnosis and treatment of kidney injury and acute kidneyinjury (AKI).

Acute ischemic injuries, such as acute kidney injury (AKI) is associatedwith high morbidity and mortality. The lack of sensitive and specificinjury biomarkers has greatly impeded the development of therapeuticstrategies to improve outcomes of ischemic injuries, e.g., AKI. Thediagnostic approach to AKI has stagnated and relies upon the biomarkers,such as, BUN, creatinine, KIM-1 and urine output, however, suchbiomarkers are inadequate to diagnose AKI as they do not directlyreflect cell injury but rather delayed functional consequences of theinjury. This has greatly impeded therapeutic innovation.

In particular, the inventors have discovered that the nucleophosmin(NPM) polypeptide, undergoes 5 different phosphorylation events duringischemia and hypoxic stress, and the detection of specificphosphorylation events of NPM in a biological sample obtained from asubject can be used to identify a subject with ischemic injury, such askidney injury, including but not limited to, acute kidney injury (AKI).Additionally, agents which inhibit the function of the NPM polypeptide,including inhibiting the association of the stress-inducedphosphorylation changes of NPM, or its subsequent association with Bax,are effective treatments for ischemia and kidney injury, including AKI.

The biologic behavior and toxicity of NPM was assessed using phospho-NPMmutant proteins that either mimic stress-induced or normal NPMphosphorylation. NPM Peptides were shown to interfere with NPM functionand demonstrate that inhibiting NPM function, in particular, inhibitingNPM from complexing with Bax, can be used to treat kidney injury and AKIeven after injury has occurred. Moreover, the inventors discovered thatwithin hours of stress, virtually identical phosphorylation changes weredetected, for example, at distinct serine/threonine sites in NPMharvested from primary renal cells in vitro, and from kidney tissue andurine in vivo. A phosphomimic NPM protein that replicatedphosphorylation under stress localized to the cytosol, formed monomersthat interacted with Bax, a cell death protein, and coaccumulated withBax in isolated mitochondria, and significantly increased cell deathafter stress. In contrast, wild-type NPM or a phosphomimic NPM with anormal phosphorylation configuration did not. Herein, the inventorsdemonstrate, with three exemplary renal targeted peptides referred toherein as “NPM peptides”, interfere with NPM at distinct functionalsites and significantly protected cells, e.g., renal cells against celldeath, and significantly, demonstrate in vivo evidence that a singledose of one peptide administered several hours after ischemia that wouldbe lethal in untreated mice significantly reduced AKI severity andimproved survival. As such, the inventors demonstrate thatphosphorylation and/or unphosphorylation at specific sites on the NPMpolypeptide serve as a potential early marker of ischemic stress,including kidney injury and ischemic AKI that links early diagnosis witheffective therapeutic interventions.

In particular, the present invention is based on, in part, the discoverythat phosphorylated NPM is both a marker of acute renal cell injury andan effective target for a range of inhibitors, herein referred to NPMinhibitory agents, such as, e.g., NPM inhibitory peptide therapeutics,siRNA and neutralizaing antibodies designed to inhibit the Bax-NPMcomplex, or its formation, and ameliorate NPM-Bax-mediated tissueinjury. The inventors have discovered that virtually identicalstress-induced, site-specific phosphorylation changes (i.e.,phosphorylation and dephosphorylation events) render NPM toxic inisolated murine and human renal cells in vitro, as well as fresh kidneytissue and urine in vivo. Furthermore, inhibiting the NPM-Bax-mediatedrenal cell death and AKI using, for example, NPM inhibitor peptides canbe used to treat or prevent kidney injury and/or AKI. In particular, theinventors have discovered that detection of the phosphorylation statusof NPM, for example, differential NPM phosphorylation, can be coupledwith NPM peptides as disclosed herein, and can be an effective AKItherapeutic treatment, even after the insult has occurred.

In particular, the inventors have discovered stress-induced differentialphosphorylation of nucleophosmin (NPM), a chaperone of the apoptoticfactor Bax, finding a virtually identical NPM stress-inducedphosphorylation pattern in mouse and human primary renal cells, freshkidney tissue, and urine within hours of injury. An NPM mimetic proteinthat replicates this stress-induced differential phosphorylation (butnot a mimetic with a normal phosphorylation state) is toxic to renalcells. The inventors have demonstrated that administering, for example atargeted NPM inhibitor peptides designed to reduce NPM toxicity, evenhours after a typically lethal ischemic insult, improves cell and animalsurvival. Accordingly, the inventors have demonstrated thatstress-induced NPM phosphorylation is a significant contributor to renalcell death in human AKI and identification of specific ischemic-inducedNPM phosphorylation changes can be used in early diagnosis andmanagement of ischemic injuries, e.g., ischemic kidney injuries.Moreover, administration of peptides or agents that inhibit thestress-induced NPM phosphorylation can be used to treat, or preventdeath or injury to renal cells in subjects with AKI, or after ischemicinsult (including hypoxia and the like).

Accordingly, one aspect of the technology described herein relates to adiagnostic assay, method and composition to assess if a subject has AKI,wherein the method comprises assessing the phosphorylation status of theNPM polypeptide, and detecting a change of at least one of thephosphorylation sites as follows: T86, S88, T95, T234 or S242. Inparticular, under normal conditions (e.g., non-stress conditions) T86,S88, T95 of the NPM polypeptide are unphosphorylated, and becomephosphorylated under stressful conditions to become phospho-T86,phospho-S88, phospho-T95. In contrast, under normal conditions (e.g.,non-stress conditions) T234 and S242 of the NPM polypeptide arephosphorylated, and become dephosphorylated under stressful conditions.That is, under normal conditions, the phosphorylation state of the NPMpolypeptide is T86, S88, T95, phospho-T234, phospho-S242, and understressful conditions, or after AKI, the phosphorylation state of the NPMpolypeptide is phospho-T86, phospho-S88, phospho-T95, T234, S242.

Accordingly, in some embodiments, an assay can comprise a method todetect at least one or more of: phospho-T86, phospho-S88, phospho-T95,unphosporylated-T234 and unphosporylated-S242 of a NPM polypeptide in abiological sample obtained from a subject, wherein detection of at leastone, or at least 2, or at least 3 or at least 4 of the abovephosphorylation states of NPM polypeptide identifies a subject withkidney injury or AKI. Detection of the phosphorylation states can be byany means, e.g., mass spectrometry, antibodies or antibody fragments,including but not limited to, pan-specific phospho-Ser (anti-pSer) orphospho-Thr (anti-pThr) antibodies, as well as phospho-specificantibodies, e.g., anti-phospho-T86, anti-phospho-S88, anti-phospho-T95,anti-phospho-T234 and anti-phospho-S242 antibodies, or antibodyfragments or antigen binding fragments thereof. In one embodiment, onelooks for a change in at least one normally unphosphorylated amino acidand one normally phosphorylated amino acid.

Exemplary biological samples include, but are not limited to, a kidneybiopsy sample, serum, blood, plasma and urine. Additionally, theinventors have also discovered that interruption of the stress-inducedNPM phosphorylation events, e.g., using one of three different blockingpeptides, can decrease cell death in the kidney due to metabolic stress(including ischemic stress and hypoxic stress), and can be used astreatment, including therapeutic treatment to treat a subject with AKI,or alternatively, as a prophylactic treatment to prevent the subjectdeveloping AKI.

Another aspect of the technology described herein relates to a method totreat a subject any of: acute kidney injury (AKI) or ischemia,comprising administering a subject a composition comprising at least onepeptide selected from the group consisting of: TVTIFVAGVLTASLTIWKKMG(SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (peptide #2) (SEQ ID NO: 2)and ESFKKQEKTPKTPKGPSSVEDIKAK (peptide #3) (SEQ ID NO: 3), or a peptidewith at least 85% or 90% or 95% sequence identity to any of SEQ ID NO:1-3. In some embodiments, the peptides of SEQ ID NO: 1-3 are conjugatedor attached to renal targeting moieties or peptides, including renaltargeting nuclear localization sequences (NLS) and/or Cell penetratingpeptides (referred to as “CPP”) as described herein, so the peptidestarget the kidney.

One aspect described herein is directed to a method of treating kidneyinjury or acute kidney injury (AKI) in a subject in need thereof, themethod comprising (a) administering a treatment for kidney injury or AKIto a subject who has first been determined to have in a biologicalsample obtained from the subject, at least one of:

-   -   i. phosphorylation of at least one of serine or threonine        residue selected from: T86, S88, or T95 of the nucleophosmin        (NPM) polypeptide; and    -   ii. absence of phosphorylation of at least one of at least one        serine or threonine residue selected from T234 or S242 on a        nucleophosmin (NPM) polypeptide,

wherein the treatment for kidney injury or AKI is an inhibitor of theformation of a Bax-NPM complex or is an inhibitor of a NPM polypeptide,wherein the NPM polypeptide comprises at the phosphorylation of at leastone of serine or threonine residue selected from: T86, S88, or T95, andabsence of phosphorylation of at least one of at least one serine orthreonine residue selected from T234 or S242.

In some embodiments, an inhibitor of the NPM polypeptide is a NPMpeptide, siRNA or antibody, and can in some embodiments, be an NPMinhibitor peptide. Exemplary NPM inhibitor peptides can be selected froma peptide comprising the amino acid sequences of: TVTIFVAGVLTASLTIWKKMG(SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ ID NO: 2) andESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3), or peptide with at least 85%sequence identity to any of SEQ ID NO: 1-3.

One aspect described herein is directed a method of treating a subjectwith kidney injury, ischemia, or a subject after an ischemic injury, themethod comprising administering to a subject a composition comprising atleast one peptide comprising the amino acid sequences of:TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ IDNO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3), or peptide with atleast 85% sequence identity to any of SEQ ID NO: 1-3.

Another aspect described herein is directed a method for inhibiting theformation of a nucleosphosmin (NMP)-Bax complex, the method comprisingcontacting a cell with at least one peptide from any peptide comprisingthe amino acid sequences of: TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1);TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ ID NO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK(SEQ ID NO: 3), or peptide with at least 85% or 90% or 95% sequenceidentity to any of SEQ ID NO: 1-3.

Another aspect described herein is directed a method for inhibitingstress induced cell death, the method comprising the method comprisingcontacting a cell with at least one peptide from any peptide comprisingthe amino acid sequences of: TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1);TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ ID NO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK(SEQ ID NO: 3), or peptide with at least 85% sequence identity to any ofSEQ ID NO: 1-3.

Another aspect described herein is directed a method to inhibitnucleosphosmin (NMP) forming a complex with Bax, the method comprisingcontacting a cell with an agent which inhibits the phosphorylation of atleast one of: T86, S88, T95 of the NMP polypeptide, and/or inhibits thedephsporylation of at least one of: T232 or S240 of the NMP polypeptide,thereby inhibiting the formation of a NMP-Bax complex.

Another aspect relates to method for inhibiting stress induced celldeath, the method comprising contacting a cell with an agent whichinhibits the phosphorylation of at least one of: T86, S88, T95 of theNMP polypeptide, and/or inhibits the dephsporylation of at least one of:T232 or S240 of the NMP polypeptide, thereby inhibiting the formation ofa NMP-Bax complex.

In all aspects herein, the methods can be used in the treatment of asubject with kidney injury, ischemia, or a subject after an ischemicinjury.

In all aspects herein, an NPM inhibitor agent is at least one peptidefrom any peptide comprising the amino acid sequences of:TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ IDNO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3), or peptide with atleast 85% sequence identity to any of SEQ ID NO: 1-3. In someembodiments, a peptide is fused to a renal targeting nuclearlocalization sequence (NSL). In some embodiments, a peptide isadministered within 48 hours of an ischemic event or ischemic injury.The method of claim 13, wherein the peptide is administered within 12hours of an ischemic event.

In all aspects herein, a kidney injury is selected from the groupconsisting of: injury to the proximal tubule of the kidney; acute kidneyinjury (AKI); chronic kidney disease (CKD); early kidney injury whichwill progress into chronic kidney disease (CKD).

In all aspects herein, ischemia is due to ischemic stress, hypoxicstress or metabolic stress, e.g., wherein the subject has ischemia inany tissue, such as, e.g, ischemia to any one or more of kidney, brain,muscle, liver, intestines or heart.

Another aspect described herein relates to diagnostic methods. In oneembodiment, a method for treating kidney injury or acute kidney injury(AKI) in a subject is described, the method comprising: (a) firstdetecting, in a biological sample obtained from the subject, at leastone of:

-   -   i. the presence of phosphorylation of at least one of serine or        threonine residue selected from: T86, S88, or T95 of the        nucleophosmin (NPM) polypeptide; or    -   ii. the absence of phosphorylation of at least one of at least        one serine or threonine residue selected from T234 or S242 on a        nucleophosmin (NPM) polypeptide; and        and (b) administering to the subject a pharmaceutical        composition comprising at least one peptide from any peptide        comprising the amino acid sequences of: TVTIFVAGVLTASLTIWKKMG        (SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ ID NO: 2) and        ESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3) or peptide with at        least 85% sequence identity to any of SEQ ID NO: 1-3, when there        is the presence of phosphorylation of at least one of serine or        threonine residue selected from T86, S88, or T95 of the NPM        polypeptide, or when there is the absence of phosphorylation on        serine residues T234 or S242 of the NPM polypeptide.

In some embodiments, the treatment for kidney injury or AKI isadministering an agent which inhibits the phosphorylation of at leastone of: T86, S88, T95 of the NMP polypeptide, and/or inhibits thedephsporylation of at least one of: T232 or S240 of the NMP polypeptide,thereby inhibiting the formation of a NMP-Bax complex.

Another aspect described herein relates to a method of determining if asubject has kidney injury or acute kidney injury (AKI), the methodcomprising:

-   -   a. using an assay to detect at least on of:        -   i. the presence of phosphorylation of at least one of serine            or threonine residue selected from: T86, S88, or T95 of the            nucleophosmin (NPM) polypeptide in a biological sample            obtained from a subject;        -   ii. the absence of phosphorylation of at least one of at            least one serine or threonine residue selected from T234 or            S242 on a nucleophosmin (NPM) polypeptide; and    -   b. selecting the subject as having kidney injury or acute kidney        injury (AKI), if the subject has the presence of phosphorylation        of at least one of: T86, S88, or T95 of the nucleophosmin (NPM)        polypeptide is detected, or the absence of phosphorylation of at        least one of at least one serine or threonine residue selected        from T234 or S242 on a nucleophosmin (NPM) polypeptide, or both,        and    -   c. administering an effective amount of a treatment for kidney        injury or AKI to the subject diagnosed in step (b).

In some embodiments of the methods described herein, a subject isdiagnosed with kidney injury or acute kidney injury (AKI) when thepresence of phosphorylation of at least two of: T86, S88, or T95 of thenucleophosmin (NPM) polypeptide is detected, or when the presence ofphosphorylation of residues T86, S88, and T95 of the nucleophosmin (NPM)polypeptide is detected, or when the absence of phosphorylation ofserine residues T234 and S242 of the nucleophosmin (NPM) polypeptide isdetected, or when the presence of phosphorylation of residues of atleast one of T86, S88, and T95 of the nucleophosmin (NPM) polypeptide isdetected and the absence of phosphorylation of at least one of serineresidues T234 and S242 of the nucleophosmin (NPM) polypeptide isdetected.

In some embodiments of the methods described herein, an effective amountof a treatment for kidney injury or AKI is administering to the subjectis an agent which inhibits the phosphorylation of at least one of: T86,S88, T95 of the NMP polypeptide, and/or inhibits the dephsporylation ofat least one of: T232 or S240 of the NMP polypeptide, thereby inhibitingthe formation of a NMP-Bax complex.

In some embodiments of the methods described herein, an effective amountof a treatment for kidney injury or AKI is administering to the subjecta composition comprising at least one peptide from any peptidecomprising the amino acid sequences of: TVTIFVAGVLTASLTIWKKMG (SEQ IDNO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ ID NO: 2) andESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3), or peptide with at least 85%sequence identity to any of SEQ ID NO: 1-3.

Another aspect described herein relates to a method comprising obtaininga biological sample from a subject, and measuring for the presence ofphosphorylation of at least one serine or threonine residue on thenucleophosmin (NPM) polypeptide at residues T86, S88, or T95, ormeasuring the absence of phosphorylation of at least one serine orthreonine residue T234 and S242 on the nucleophosmin (NPM) polypeptide,or both. In some embodiments, the method further comprises detecting thepresence of phosphorylation of at least two residues on the NMPpolypeptide at residues T86, S88, or T95, or detecting the presence ofphosphorylation at residues T86, S88, or T95 on the NMP polypeptide, orfurther comprises detecting the absence of phosphorylation at residuesT234 or S242 on the NMP polypeptide.

Another aspect described herein relates to a method comprising obtaininga biological sample from a subject, and measuring for the presence ofphosphorylation of at least one serine or threonine residue on thenucleophosmin (NPM) polypeptide at residues T86, S88, or T95, andmeasuring the absence of phosphorylation of at least one serine orthreonine residue T234 and S242 on the nucleophosmin (NPM) polypeptide.

In some embodiments of the methods described herein, the subject is amammal, e.g., a human. In some embodiments, the subject is at risk ofdeveloping renal injury or acute kidney injury (AKI).

In some embodiments of the methods described herein, the biologicalsample is a urine sample or blood sample, e.g., a blood sample selectedfrom any of the group consisting of; a whole blood sample a plasmasample, a serum sample or a fractionated blood sample.

In some embodiments of the methods described herein, the presence ofphosphorylation of at least one residues on the NMP polypeptide atresidues T86, S88, or T95 is determined by detecting the presence ofbinding of a phosphorylation specific antibody that preferentially bindto any one of: phospho-T86, phopho-S88 or phospho-T95 on the NPMpolypeptide.

In some embodiments of the methods described herein, the absence ofphosphorylation of at least one serine or threonine residue T234 andS242 on the nucleophosmin (NPM) polypeptide is determined by detectingthe absence of binding of a phosphorylation specific antibody thatpreferentially bind to any one of: phospho-T234 or phopho-S242 on theNPM polypeptide.

In some embodiments of the methods described herein, the presence ofphosphorylation of at least one residues on the NMP polypeptide atresidues T86, S88, or T95 is determined by the presence of binding withat least one of:

-   -   a. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T86 on the        NMP polypeptide;    -   b. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S886 on the        NMP polypeptide; or    -   c. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T95 on the        NMP polypeptide.

In some embodiments of the methods described herein, the absence ofphosphorylation of at least one serine or threonine residue selectedfrom T234 and S242 on the NPM polypeptide is determined by the absenceof binding with at least one of:

-   -   a. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T234 on the        NMP polypeptide; or    -   b. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S242 on the        NMP polypeptide.

In some embodiments of the methods described herein, a site-specificphosphorylation specific antibody or antigen-binding fragment thereofare selected from the group consisting of: a polyclonal antibody, achimeric antibody, an Fab antigen-binding fragment thereof, fragment, anF(ab′)2 antigen-binding fragment thereof, an Fab′ antigen-bindingfragment thereof, an Fsc antigen-binding fragment thereof, or an Fvantigen-binding fragment thereof. In some embodiments, a site-specificphosphorylation specific antibody or antigen-binding fragment thereof isselected from the group consisting of: a recombinant antibody, achimeric antibody, a tribody, a midibody or a monoclonoal antibody. Insome embodiments, a phosphorylation specific antibody or antigen-bindingfragment thereof is a humanized antibody or a human antibody. In someembodiments, antibodies, including but not limited to, a phosphorylationspecific antibody or antigen-binding fragment thereof is immobilized on,or attached to, the surface of a solid support, including, but notlimited to a solid support surface in the format of a dipstick, a teststrip, paper-based assay, a latex bead, a microsphere, or a multi-wellplate.

In some embodiments, an antibody, including, but not limited to,phosphorylation specific antibody or antigen-binding fragment thereofcomprise a detectable label, or can be bound by a secondary agent whichcomprises a detectable label.

In some embodiments of the methods described herein, detecting thepresence of phosphorylation of at least one serine or threonine residueon the nucleophosmin (NPM) polypeptide at residues T86, S88, or T95, ordetecting the absence of phosphorylation of at least one serine orthreonine residue T234 and S242 on the nucleophosmin (NPM) polypeptide,or both uses an immunoassay selected from the group consisting of: anELISA assay, multiplex bead assay, dipstick assay, Western blotanalysis, radioimmunoassay (RIA), Immunoradiometric assay (IRMA),chemiluminescent immunoassays, a fluorescence antibody method, passivehaemagglutination.

Another aspect relates to a kit comprising a solid support and affixedto the solid support at least one antibody selected from:

-   -   a. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T86 on the        NMP polypeptide,    -   b. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S886 on the        NMP polypeptide,    -   c. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T95 on the        NMP polypeptide,    -   d. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T234 on the        NMP polypeptide; or    -   e. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S242 on the        NMP polypeptide,    -   f. a specific antibody or antigen-binding fragment thereof that        specifically binds to non-phosphorylated T234 (nT234) on the NMP        polypeptide; or    -   g. a specific antibody or antigen-binding fragment thereof that        specifically binds to non-phosphorylated S242 (nS242) on the NMP        polypeptide, and    -   and an antibody that specifically binds to total NMP        polypeptide, and one or more detection means to detect one or        more of the antibodies.

In some embodiments, the kit comprises, affixed to the solid support, atleast one antibody selected from:

-   -   a. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T86 on the        NMP polypeptide,    -   b. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S886 on the        NMP polypeptide,    -   c. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T95 on the        NMP polypeptide,

at least one antibody selected from:

-   -   d. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T234 on the        NMP polypeptide; or    -   e. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S242 on the        NMP polypeptide.

In alternative embodiments, the kit comprises, affixed to the solidsupport:

at least one antibody selected from:

-   -   f. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T86 on the        NMP polypeptide,    -   g. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-S886 on the        NMP polypeptide,    -   h. a phosphorylation specific antibody or antigen-binding        fragment thereof that specifically binds to phospho-T95 on the        NMP polypeptide,

at least one antibody selected from:

-   -   a. a specific antibody or antigen-binding fragment thereof that        specifically binds to non-phosphorylated T234 (nT234) on the NMP        polypeptide; or    -   b. a specific antibody or antigen-binding fragment thereof that        specifically binds to non-phosphorylated S242 (nS242) on the NMP        polypeptide.

In some embodiments, the kit can comprise at least 2, or at least 3, orat least 4, or at least 5 specific antibody or antigen-binding fragmentthereof described herein.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show stress causes cytosolic nucleophosmin (NPM)translocation. FIG. 1A shows quantitative assessment of NPM in thenuclear and cytosolic cell fractions of primary human proximal tubuleepithelial cells (PTECs) subjected to 60 minutes ATP depletion;identical amounts of proteins were loaded in each lane, anddensitometric analysis was used to assess the relative amount of NPMpresent in the cytosolic and noncytosolic fractions before (Base) andafter ATP depletion (ATP Depl; n=3). FIG. 1B shows NPM accumulationdetected in the cytosolic fraction of renal cortical homogenatesharvested from paired donor kidneys with either normal perfusion(normal) or perfusion pump failure (ischemic; results represent fourkidneys from two human donors), FIG. 1C shows NPM accumulation inprimary murine or human PTECs subjected to ATP depletion 360 minutes,and FIG. 1D shows NPM accumulation in murine and human PTECs subjectedto 70 minutes of hypoxia. In B-D, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) serves as a loading control; densitometryrepresents three independent experiments in each study. Cytosolicfractions were harvested using digitonin (see Methods); noncytosolicextracts were harvested by exposing cells to RIPA buffer afterextracting the cytosolic fraction. CTL, control; RDU, relative densityunit

FIGS. 2A-2D show mass spectrometry reveals consistent differentialphosphorylation changes in NPM harvested from in renal cells, kidneytissue, and urine. Representative tracings used to perform differentialphosphoproteomic analysis of purified NPM harvested from the renalcortex of an ischemic human kidney are shown. FIG. 2A shows liquidchromatography/mass spectrometry base peak peptide profile of an NPMamino acid fragment. FIG. 2B and FIG. 2C show liquidchromatography/tandem mass spectrometry (MS/MS) peptidecollision-induced dissociation fragment ions providing de novo peptidesequence and specific serine phosphorylation sites represented by lossof 2167 D (S-87+P-80 D). FIG. 2D shows a summary of five differentiallyNPM-phosphorylated sites altered in a virtually identical manner byischemic stress in primary murine cells or human proximal tubuleepithelial cells (PTECs), murine kidney (mKdy), and human kidney (hKdy)versus control (CTL; murine PTEC, mKdy, and hKdy controls were identicaland are shown together). Murine AKI urine (mAKI), human AKI urine(hAKI), and human non-AKI urine (hNonAKI) are also shown.Phosphorylation absent is indicated by a cross; phosphorylation presentis indicated by a star. Asterisks show homology between human NPM andmurine NPM at residues T234/T232 and S242/S240, respectively. The kinaseconsensus sequences for each of the five phosphorylation NPM sites areidentical between these two species. N/D, not detected.

FIG. 3A-3C shows renal ischemia increases urinary nucleophosmin (NPM).FIG. 3A shows marked accumulation of total NPM in murine urine wasdetected by dot blot analysis 6 and 12 hours after 25 minutes oftransient bilateral renal ischemia, but it disappeared within 48 hourspostischemia. In contrast, no urinary NPM was detected at the same timepoints in sham-operated animals. FIG. 3B is immunohistochemistry showingNPM leakage into the urine in ischemic human kidney after AKI. *identifies that NPM is localized in the urinary space, and arrowsindicate medullary tubule cell nuclei. (400× magnification). FIG. 3Cshows that ischemic AKI causes NPM re-distribution. Fresh frozen kidneybiopsy sections were double immunostained with a NPM antibody anddetected with a fluorescent goat anti-mouse IgG antibody (red) andHoechst dye #33342. The left panel shows the NPM localization from apatient with no AKI showing nucleolar localization of NPM distributiontypical for normal renal cells (white arrows), whereas the right panelshows ischemic AKI showing NPM diffusely distributed in virtually allcells and the nuclei are negative as reported in ischemic PTEC, andindicates luminal NPM staining (i.e., NPM is localized in the urinaryspace of kidneys from a subject with AKI).

FIG. 4A-4B shows differentially phosphorylated nucleophosmin (NPM) siteslocalize near its structural and functional domains. FIG. 4A shows thelocation of NPM phosphosites detected in postischemic proximal tubuleepithelial cells (PTECs), kidney tissue, and urine. Therapeutic peptide#2 (representing NPM amino acids 78-103 (SEQ ID NO: 2)) and 3(representing NPM amino acids 226-246 (SEQ ID NO: 3)) were designed tointerfere with NPM functions at the indicated phospho-sitesdifferentially phosphorylated during ischemic stress. Site-specificamino acid substitutions were made to render each site eitherconstitutively phosphorylated (E substituted for S or T) orconstitutively dephosphorylated (A substituted for S or T) to replicatethe phosphorylation status of wild-type NPM under resting or ischemicstress conditions (as described in FIG. 2D). FIG. 4B shows a table ofall possible combinations of different NPM-phospho-mimics (32 total).Amino acid substitutions mimic each site in either is constitutivelyphosphorylated state (S or T to E) or constitutively de-phosphorylatedstate (S or T to A). Therapeutic peptides #2 (SEQ ID NO: 2) (NPM(78-103)) and peptide #3 (SEQ ID NO: 3) (NPM(226-246) replicate nativeNPM consensus sequences. Peptide #10 is the “normal NPM mimic”, andPeptide #22 is the “stress NPM mimetic”.

FIGS. 5A-5E shows phosphomimic nucleophosmin (NPM) proteins thatreplicate stress-induced phosphorylation changes regulate NPM toxicity.The biologic behavior of nucleophosmin (NPM) phosphomimic proteinsassessed using functional bioassays performed in primary murine proximaltubule epithelial cells (PTECs) that express flag-tagged wild type (WT),a normal (Nor-M or NrNPM) NPM mimic protein, or a stress (Stre-M, StNPM,or Stress-M) NPM mimic protein that replicates differential NPMphosphorylation. FIG. 5A shows NPM localization in intact renal cells inthe absence of stress. FIG. 5B shows NPM oligomers and monomers detectedin lysates harvested from nonstressed cells in a native gel; total NPMcontent is unchanged in each lane, and the increase in monomeric NPM isaccompanied by a reciprocal decrease in the oligomeric form of NPM. FIG.5C shows NPM-Bax complex formation assessed after immunoprecipitation(IP) of flagtagged NPM followed by immunoblot (IB) of conformationallyactivated Bax (Bax 6A7) after ischemic stress; input protein amountswere similar in each sample. FIG. 5D shows accumulation of NPM and 6A7Bax in isolated PTEC mitochondria after ischemic stress; VDAC, amitochondrial membrane protein, serves as loading control. FIG. 5E showscell survival after 60 minutes ATP depletion (n=6). EV, empty sgOptivector; RDU, relative density unit. *P, 0.05 for Nor-M and Stress-M NPMphosphomimics versus control (CTL).

FIG. 6 shows nucleophosmin (NPM) suppression increases proximal tubuleepithelial cell (PTEC) survival after ischemic stress. An inducibleclustered regularly interspaced short palindromic repeats-based systemsuppressed NPM expression after doxycycline exposure (Cri; 2 mg/ml 372hours; inset) versus empty sgOpti vector (EV; inset). NPM suppressionsignificantly improved human PTEC survival after 60 minutes of ATPdepletion. CRISPRi, clustered regularly interspaced short palindromicrepeats interference. *P, 0.05 EV versus Cri (n=4).

FIG. 7 shows therapeutic peptides improve renal cell survival afterischemic stress. Peptides designed to reduce nucleophosmin (NPM)-Baxcomplex formation (Bax peptide) (SEQ ID NO: 1) or interfere withphosphorylation changes that regulate NPM toxicity (peptides #2 (SEQ IDNO: 2) and #3 (SEQ ID NO: 3)) significantly improve cell survival afterischemic stress (n=4; upper panel). Peptide 2 replicates regulatoryphosphorylation sites located at the amino terminus (T86, S88, and T95).Peptide 3 replicates regulatory phosphorylation sites located at thecarboxy terminus (T234 and S242). The amino acid sequences of each ofthe three peptides designed to interfere with NPM function are shown(lower panel). *P, 0.05 versus control peptide (CTL).

FIGS. 8A-8B show therapeutic peptide treats ischemic AKI. FIG. 8A showsthat after 28 minutes of bilateral renal pedicle clamping, a single dose(100 μg/g body weight) of NPM-Bax blocking peptide (Peptide #1) wasintravenously administered to each of seven paired animal groups at 0,1, 2, 3, 4, 5, or 6 hours (n=8 animals for each group; total of 56 mice)or control (n=6 animals for each group; total of 42 mice). Blockingpeptide administered within 3 hours postischemia significantly improvedrenal function (i.e., lowered serum BUN) on days 1-7 and dramaticallyimproved animal survival (P, 0.05 versus control). The magnitude ofrenoprotection was similar with peptide given at 0, 1, and 3 hourspostischemia (P, 0.05 for 0 versus 1 or 3 hours); only data for blockingand control peptides administered 3 hours after ischemia are shown. FIG.8B shows serum creatinine on day 1 (the time of peak kidney dysfunction)was significantly reduced when the blocking peptide was administered 3hours after ischemia; *P, 0.05 versus control (CTL).

FIGS. 9A-9B shows that the NPM-Bax blocking peptide (Peptide #1)decreases nucleophosmin (NPM)-Bax interaction. FIG. 9A shows that activeBax immunoprecipitates harvested from human proximal tubule epithelialcell (PTEC) lysates after 60 minutes of ATP depletion plus 30 minutes ofrecovery. FIG. 9B shows Bax immunoprecititates from renal corticalhomogenates after ischemia in the absence of a peptide (None), a controlpeptide (CTL), or a NPM-Bax blocking peptide (Peptide #1) (Block)injected 3 hours after bilateral renal ischemia. Similar amounts of Baxwere detected in each lane, and input protein amounts for each samplewere identical. IB, immunoblot; IP, immunoprecipitation.

FIG. 10 shows five phosphorylation changes mediate NPM toxicity duringischemic stress. Based on the results of the NPM bioassays (FIG. 5),phosphorylation events at S88 and T9540,41 regulate steps 1-3 in thenucleophosmin-Bax cell death pathway. T95 phosphorylation also regulatescell death in response to chemotherapy and radiation therapy in acutemyelogenous leukemia.40,41 Release from nucleolar binding sites isassumed to occur before NPM deoligomerization or cytosolictranslocation.45,75 The function of NPM-T86, T234, and S242phosphorylation changes during ischemic cell death also occurs.

FIG. 11 shows exemplary peptides or pharmaceuticals (e.g., smallmolecules) that can be used on combination with a NPM inhibitor agent asdefined herein. These agents include geronylgeronylacetone (GGA), apotent Hsp70 inducer86, avrainvillamide, and NSC34888437.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), FieldsVirology, 6^(th) Edition, published by Lippincott Williams & Wilkins,Philadelphia, Pa., USA (2013), Knipe, D. M. and Howley, P. M. (ed.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

As used herein, “kidney injury” includes any injury to the proximaltubule of the kidney and includes, but is not limited to, acute kidneyinjury (AKI), chronic kidney disease (CKD) and kidney fibrosis.

As used herein, “acute kidney injury”, also known as “AKI” or “acuterenal failure (ARF)” or “acute kidney failure”, refers to a disease orcondition where a rapid loss of renal function occurs due to damage tothe kidneys, resulting in retention of nitrogenous (urea and creatinine)and non-nitrogenous waste products that are normally excreted by thekidney. Depending on the severity and duration of the renal dysfunction,this accumulation is accompanied by metabolic disturbances, such asmetabolic acidosis (acidification of the blood) and hyperkalaemia(elevated potassium levels), changes in body fluid balance, and effectson many other organ systems. It can be characterized by oliguria oranuria (decrease or cessation of urine production), although nonoliguricARF may occur. Acute kidney injury may be a consequence of variouscauses including a) pre-renal (causes in the blood supply), whichincludes, but is not limited to, hypovolemia or decreased blood volume,usually from shock or dehydration and fluid loss or excessive diureticsuse; hepatorenal syndrome, in which renal perfusion is compromised inliver failure; vascular problems, such as atheroembolic disease andrenal vein thrombosis, which can occur as a complication of nephroticsyndrome; infection, usually sepsis, and systemic inflammation due toinfection; severe burns; sequestration due to pericarditis andpancreatitis; and hypotension due to antihypertensives and vasodilators;b) intrinsic renal damage, which includes, but is not limited to, toxinsor medication (e.g. some NSAIDs, aminoglycoside antibiotics, iodinatedcontrast, lithium, phosphate nephropathy due to bowel preparation forcolonoscopy with sodium phosphates); rhabdomyolysis or breakdown ofmuscle tissue, where the resultant release of myoglobin in the bloodaffects the kidney, which can also be caused by injury (especially crushinjury and extensive blunt trauma), statins, stimulants and some otherdrugs; hemolysis or breakdown of red blood cells, which can be caused byvarious conditions such as sickle-cell disease, and lupus erythematosus;multiple myeloma, either due to hypercalcemia or “cast nephropathy”;acute glomerulonephritis which may be due to a variety of causes, suchas anti-glomerular basement membrane disease/Goodpasture's syndrome,Wegener's granulomatosis or acute lupus nephritis with systemic lupuserythematosus; and c) post-renal causes (obstructive causes in theurinary tract) which include, but are not limited to, medicationinterfering with normal bladder emptying (e.g. anticholinergics); benignprostatic hypertrophy or prostate cancer; kidney stones; abdominalmalignancy (e.g. ovarian cancer, colorectal cancer); obstructed urinarycatheter; or drugs that can cause crystalluria and drugs that can leadto myoglobinuria & cystitis.

As used herein, the term “kidney fibrosis” also known as “renalfibrosis” refers to any condition having kidney fibrosis as a symptom orcause of the condition, or a condition that can be worsened by thedevelopment of kidney fibrosis, or a condition the progression of whichis linked to the progression of kidney fibrosis. Kidney fibrosis is theformation of excess fibrous connective tissue in kidney characterized byglomerulosclerosis and tubulointerstitial fibrosis. The pathogenesis ofkidney fibrosis is a monotonous process that is characterized by anexcessive accumulation and deposition of extracellular matrix (ECM)components (see e.g., Y. Liu, Kidney International 2006, 69, 213-217).Kidney fibrosis can be evaluated by methods including, but not limitedto, histology, immunohistochemistry, Western blot, and real-time PCR formRNA and protein expression of extracellular matrix including collagen Iand alpha-smooth muscle actin, and activation of TGF beta/Smadsignaling. Kidney fibrosis can result from various diseases and insultsto the kidneys. Examples of such diseases and insults include chronickidney disease, metabolic syndrome, vesicoureteral reflux,tubulointerstitial renal fibrosis, diabetes (including diabeticnephropathy), and resultant glomerular nephritis (GN), including, butnot limited to, focal segmental glomerulosclerosis and membranousglomerulonephritis, mesangiocapillary GN. Since kidney fibrosis isassociated with loss of blood vessels, this results in secondaryischemia which can also result in glomerulare disease with loss ofglomerular function. Regardless of the primary cause, insults to thekidneys may result in kidney fibrosis and the concomitant loss of kidneyfunction. (Schena, F. and Gesualdo, L., Pathogenic Mechanisms ofDiabetic Nephropathy, J. Am. Soc. Nephrol., 16: S30-33 (2005);Whaley-Connell, A., and Sower, J R., Chronic Kidney Disease and theCardiometabolic Syndrome, J. Clin. Hypert., 8(8): 546-48 (2006)).Conditions associated with kidney fibrosis include, but are not limitedto, diabetic nephropathy, chronic kidney disease, end-stage renaldisease, systemic lupus erythematosis, vasculitis, IgA nephropathy,other autoimmune diseases, paraprotein diseases, diabetes. In someembodiments, a condition associated with kidney fibrosis results frompersistent KIM-1 expression in kidney cells. Renal Fibrosis has threestages which are inflammation reaction stage, formation of fibrosisstage and cicatricial stage respectively. Symptoms vary depending on thestage. There are no obvious symptoms in the inflammation reaction stage.In the formation stage, symptoms occur such as frequent night urine,high potassium, high blood pressure and itchy skin and so on. In thecicatricial stage, renal failure may occur.

As used herein, a “subject” refers to a mammal, preferably a human. Theterm “individual”, “subject”, and “patient” are used interchangeablyherein, and refer to an animal, for example a mammal, such as a human.The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited: to humans, non-humanprimates such as apes, monkeys, orangutans, and chimpanzees; canids suchas dogs and wolves; felids such as cats, lions, and tigers; equids suchas horses, donkeys, and zebras; food animals such as cows, pigs, andsheep; ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears, as well as commercial livestock andcompanion animals.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “sample” or “biological sample” refers to asample of tissue or fluid obtained from the subject. In someembodiments, the term “blood samples” include, but are not limited to,whole blood, serum or plasma. In some embodiments, the whole bloodsample is further processed into serum or plasma samples. The term alsoincludes a mixture of the above-mentioned samples. The term “sample”also includes untreated or pretreated (or pre-processed) samples. Insome embodiments, a sample can comprise one or more cells from thesubject. In some embodiments, a sample is taken from a human subject,and in alternative embodiments the sample is taken from any mammal, suchas rodents, animal models of diseases, commercial animals, companionanimals, dogs, cats, sheep, cattle, and pigs, etc. The sample can bepretreated as necessary for storage or preservation, by dilution in anappropriate buffer solution or concentrated, if desired. Any of a numberof standard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used. The sample can in certain circumstances be stored for use priorto use in the assays as disclosed herein. Such storage can be at +4° C.or frozen, for example at −20° C. or −80° C. In some embodiments, thebiological sample is cryopreserved. In some embodiments, the biologicalsample is “fixed” or otherwise preserved as to preserve thephosphorylation status of the biological sample, and/or treated withphosphatase inhibitors and/or kinase inhibitors to preventdephosphorylation or phosphorylation events from occurring afterobtaining the biological sample from the subject. In some embodiments,the biological sample is immediately frozen in liquid nitrogen or dryice to preserve the phosphorylation status of the biological sample.

As used herein, the term “biomarker” or refers to a phenotype of apolypeptide expressed endogenously in an individual or found orsequestered in a sample from an individual. The term “acute kidneyinjury biomarker” is used throughout the specification as an example ofa type of biomarker useful with the methods described herein. Acutekidney injury and pyelonephritis are examples of conditions associatedwith a biomarker as the term “biomarker” is used herein. A biomarker oracute kidney injury biomarker can include the NPM polypeptide with atleast 1, 2, 3, 4 or all 5 of the phosphorylation states of: pT86, pS88,pT95, nT234, nS242. The phosphorylation status of the NPM polypeptide asa biomarker useful for diagnosing AKI also encompasses domains orfragments of NPM polypeptide, as well as species, variants, homologues,allelic forms, mutant forms, and equivalents of NPM polypeptide. In someembodiments, the NPM polypeptide is human NPM polypeptide.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includessingle, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNAhybrids, or a polymer including purine and pyrimidine bases or othernatural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. “Oligonucleotide” generally refers topolynucleotides of between about 5 and about 100 nucleotides of single-or double-stranded DNA. However, for the purposes of this disclosure,there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as “oligomers” or “oligos” and may beisolated from genes, or chemically synthesized by methods known in theart. The terms “polynucleotide” and “nucleic acid” should be understoodto include, as applicable to the embodiments being described,single-stranded (such as sense or antisense) and double-strandedpolynucleotides.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature orwhich is synthetic. The term nucleic acid construct is synonymous withthe term “expression cassette” when the nucleic acid construct containsthe control sequences required for expression of a coding sequence ofthe present disclosure. An “expression cassette” includes a DNA codingsequence operably linked to a promoter.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

A DNA sequence that “encodes” a particular RNA or protein gene productis a DNA nucleic acid sequence that is transcribed into the particularRNA and/or protein. A DNA polynucleotide may encode an RNA (mRNA) thatis translated into protein, or a DNA polynucleotide may encode an RNAthat is not translated into protein (e.g., tRNA, rRNA, or aDNA-targeting RNA; also called “non-coding” RNA or “ncRNA”).

As used herein the term “agent” refers to a protein-binding agent thatspecifically binds to a target protein and permits detection and/orquantification of phosphorylation levels, as well as proteinconcentrations, expression levels, or activity of the total protein in abiological sample. Such protein-binding agents include, but are notlimited to, small molecules, antibodies, antibody fragments (e.g.,antigen-binding fragments of antibodies), recombinant antibodies,chimeric antibodies, tribodies, midibodies, protein-binding agents,small molecules, recombinant protein, peptides, aptamers, avimers andprotein-binding derivatives or fragments thereof.

The terms “protein-binding molecule” refers to an agent or protein whichspecifically binds to a protein, such as a protein-binding moleculewhich specifically binds a NPM polypeptide or a particularphosphorylation site on the NPM polypeptide (any one or more of: pT86,pS88, pT95, nT234, nS242). Protein-binding molecules are well known inthe art, and include antibodies, protein-binding peptide and the like.The region on the protein which binds to the protein-binding molecule isreferred to as the epitope, and the protein which is bound to theprotein-binding molecule is often referred to in the art as an antigen.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bind anantigen. The terms also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms besides antibodies; including, for example, Fv,Fab, and F(ab)′2 as well as bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory (1988) andHunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporatedherein by reference). In some embodiments, antibody reagents, e.g.antibodies, monoclonal and chimeric antibodies useful in the methods asdisclosed herein can be manufactured using well-known methods, e. g., asdescribed in Howard and Kaser “Marking and Using Antibodies: A PracticalHandbook” CRC Press (2006); which is incorporated by reference herein inits entirety. Antibody fragments or antigen-binding antibody fragmentsincludes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule, and include, but are notlimited to a complementarily determining region (CDR) of a heavy orlight chain or a ligand binding portion thereof, a heavy chain or lightchain variable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, any of which can be incorporated into an antibodyof the present invention. The antibodies can be polyclonal or monoclonaland can be isolated from any suitable biological source, e.g., murine,rat, sheep and canine. Additional sources are identified infra. The term“antibody” is further intended to encompass digestion fragments,specified portions, derivatives and variants thereof, including antibodymimetics or comprising portions of antibodies that mimic the; structureand/or function of an antibody or specified fragment or portion thereof,including single chain antibodies and fragments thereof. Examples ofbinding fragments encompassed within the term “antigen binding portion”of an antibody include a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH, domains; a F(ab′) 2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; a Ed fragment consisting of the VH and CH, domains; aFv fragment consisting of the VL and VH domains of a single arm of anantibody, a dAb fragment (Ward et al. (1989) Nature 341:544-546), whichconsists of a VH domain; and an isolated complementarily determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv)). Bird et al.(1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883. Single chain antibodies are also intended to beencompassed within the term “fragment of an antibody.” Any of theabove-noted antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for binding specificity and neutralization activity in the samemanner as are intact antibodies.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein to refer to one or more fragments of afull length antibody that retain the ability to specifically bind to atarget of interest. Examples of binding fragments encompassed within theterm “antigen-binding fragment” of a full length antibody include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fabfragments linked by a disulfide bridge at the hinge region; (iii) an Fdfragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546; which isincorporated by reference herein in its entirety), which consists of aVH or VL domain; and (vi) an isolated complementarity determining region(CDR) that retains specific antigen-binding functionality. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules knownas single chain Fv (scFv). See e.g., U.S. Pat. Nos. 5,260,203,4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibodyfragments can be obtained using any appropriate technique includingconventional techniques known to those of skill in the art. The term“monospecific antibody” refers to an antibody that displays a singlebinding specificity and affinity for a particular target, e.g., epitope.This term includes a “monoclonal antibody” or “monoclonal antibodycomposition,” which as used herein refer to a preparation of antibodiesor fragments thereof of single molecular composition, irrespective ofhow the antibody was generated.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The phrase can also refer to continuous or discontinuousepitopes in which the primary sequence (i.e., the amino acid sequence)is not similar but nonetheless the epitopes are still recognized by thesame antibody.

The term “antibody variant” is intended to include antibodies producedin a species other than a mouse. It also includes antibodies containingpost translational modifications to the linear polypeptide sequence ofthe antibody or fragment. It further encompasses fully human antibodies.The term “antibody derivative” is intended to encompass molecules thatbind an epitope as defined above and which are modifications orderivatives of a native monoclonal antibody of this invention.Derivatives include, but are not limited to, for example, bispecific,multispecific, heterospecific, trispecific, tetraspecific, multispecificantibodies, diabodies, chimeric, recombinant and humanized.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the presentinvention can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in viva).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, CL, CH domains (e.g., CH1, CH2, CH3), hinge,(Via, VH)) is substantially non-immunogenic in humans, with only minorsequence changes or variations. Similarly, antibodies designated primate(monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guineapig, hamster, and the like) and other mammals designate such species,sub-genus, genus, sub-family, family specific antibodies. Further,chimeric antibodies include any combination of the above. Such changesor variations optionally and preferably retain or reduce theimmunogenicity in humans or other species relative to non-modifiedantibodies. Thus, a human antibody is distinct from a chimeric orhumanized antibody. It is pointed out that a human antibody can beproduced by a non-human animal or prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin(e.g., heavy chain and/or light chain); genes. Further, when a humanantibody is a single chain antibody, it can comprise a linker peptidethat is not found in native human antibodies. For example, an Fv cancomprise a linker peptide, such as two to about eight glycine or otheramino acid residues, which connects the variable region of the heavychain and the variable region of the light chain. Such linker peptidesare considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody can be at least about 95%, or even at leastabout 96%, or least about 97%, or least about 98%, or least about 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline immunoglobulin gene. Typically, a human antibody derivedfrom a particular human germline sequence will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody candisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable and constant regionsderived from human germline immunoglobulin sequences. The term“recombinant human antibody”, as used herein, includes all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in viva somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, can not naturally existwithin the human antibody germline repertoire in vivo. As used herein,“isotype” refers to the antibody class (e.g., IgM or IgG1) that isencoded by heavy chain constant region genes.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen-binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byRabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity (e.g., antibody or antigen-binding fragment)binds to the second, target entity (e.g., NPM polypeptide, and/or thespecific phosphorylation sites of pT86, pS88, pT95, nT234, nS242 the NPMpolypeptide) with greater specificity and affinity than it binds to athird entity which is a non-target. In some embodiments, specificbinding can refer to an affinity of the first entity for the secondtarget entity which is at least 10 times, at least 50 times, at least100 times, at least 500 times, at least 1000 times or greater than theaffinity for the third non-target entity. In particular, the terms“specifically binds,” “specific binding affinity” (or simply “specificaffinity”), and “specifically recognize,” and other related terms whenused to refer to binding between a protein and an antibody, refers to abinding reaction that is determinative of the presence of the protein inthe presence of a heterogeneous population of proteins and otherbiologies. Thus, under designated conditions, a specified antibody bindspreferentially to a particular epitope (e.g., any one of pT86, pS88,pT95, nT234, nS242 on the NPM polypeptide) and does not bind in asignificant amount to other proteins present in the sample. An antibodythat specifically binds to a protein has an association constant of atleast 10³M⁻¹ or 10⁴M⁻¹, sometimes 10⁵M⁻¹ or 10⁶M⁻¹, in other instances10⁶M⁻¹ or 10¹⁰M⁻¹, preferably 10⁸M⁻¹ to 10⁹M⁻¹, and more preferably,about 10¹⁰M⁻¹ to 10¹¹M⁻¹ or higher. Protein-binding molecules withaffinities greater than 10⁸M⁻¹ are useful in the methods of the presentinvention. A variety of immunoassay formats can be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

An “array” broadly refers to an arrangement of agents (e.g., proteins,antibodies, replicable genetic packages) in positionally distinctlocations on a substrate. In some instances the agents on the array arespatially encoded such that the identity of an agent can be determinedfrom its location on the array. A “microarray” generally refers to anarray in which detection requires the use of microscopic detection todetect complexes formed with agents on the substrate. A “location” on anarray refers to a localized area on the array surface that includesagents, each defined so that it can be distinguished from adjacentlocations (e.g., being positioned on the overall array, or having somedetectable characteristic, that allows the location to be distinguishedfrom other locations). Typically, each location includes a single typeof agent but this is not required. The location can have any convenientshape (e.g., circular, rectangular, elliptical or wedge-shaped). Thesize or area of a location can vary significantly. In some instances,the area of a location is greater than 1 cm2, such as 2 cm2, includingany area within this range. More typically, the area of the location isless than 1 cm2, in other instances less than 1 mm2, in still otherinstances less than 0.5 mm², in yet still other instances less than10,000 mm², or less than 100 mm².

A“label” refers to an agent that can be detected by using physical,chemical, optical, electromagnetic and/or other methods. Examples ofdetectable labels that can be utilized include, but are not limited to,radioisotopes, fluorophores, chromophores, mass labels, electron denseparticles, magnetic particles, spin labels, molecules that emitchemiluminescence, electrochemically active molecules, enzymes,cofactors, and enzyme substrates.

As used herein, the terms “proteins” and “polypeptides” are usedinterchangeably to designate a series of amino acid residues connectedto each other by peptide bonds between the alpha-amino and carboxygroups of adjacent residues. The terms “protein”, and “polypeptide”refer to a polymer of amino acids, including modified amino acids (e.g.,phosphorylated, glycated, glycosylated, etc.) and amino acid analogs,regardless of its size or function. “Protein” and “polypeptide” areoften used in reference to relatively large polypeptides, whereas theterm “peptide” is often used in reference to small polypeptides, butusage of these terms in the art overlaps. The terms “protein” and“polypeptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary polypeptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

The terms “disease”, “disorder”, or “condition” are used interchangeablyherein, refer to any alternation in state of the body or of some of theorgans, interrupting or disturbing the performance of the functionsand/or causing symptoms such as discomfort, dysfunction, distress, oreven death to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, affectation.

The term “cancer” or “malignancy” are used interchangeably herein,refers to diseases that are characterized by uncontrolled, abnormalgrowth of cells which results in an increase in a particular cell typeor increase in a tissue growth or tissue mass. Cancer cells can spreadlocally or through the bloodstream and lymphatic system to other partsof the body. The term is also intended to include any disease of anorgan or tissue in mammals characterized by poorly controlled oruncontrolled multiplication of normal or abnormal cells in that tissueand its effect on the body as a whole. Cancer diseases within the scopeof the definition comprise benign neoplasms, dysplasias, hyperplasias aswell as neoplasms showing metastatic growth or any other transformationslike e.g. leukoplakias which often precede a breakout of cancer.

As used herein, the term “tumor” refers to a mass of transformed cellsthat are characterized, at least in part, by containing angiogenicvasculature. The transformed cells are characterized by neoplasticuncontrolled cell multiplication which is rapid and continues even afterthe stimuli that initiated the new growth has ceased. The term “tumor”is used broadly to include the tumor parenchymal cells as well as thesupporting stroma, including the angiogenic blood vessels thatinfiltrate the tumor parenchymal cell mass. Although a tumor generallyis a malignant tumor, i.e., a cancer having the ability to metastasize(i.e. a metastatic tumor), a tumor also can be nonmalignant (i.e.non-metastatic tumor). Tumors are hallmarks of cancer, a neoplasticdisease the natural course of which is fatal. Cancer cells exhibit theproperties of invasion and metastasis and are highly anaplastic.

As used herein, the term “metastases” or “metastatic tumor” refers to asecondary tumor that grows separately elsewhere in the body from theprimary tumor and has arisen from detached, transported cells, whereinthe primary tumor is a solid tumor. The primary tumor, as used herein,refers to a tumor that originated in the location or organ in which itis present and did not metastasize to that location from anotherlocation. As used herein, a “malignant tumor” is one having theproperties of invasion and metastasis and showing a high degree ofanaplasia. Anaplasia is the reversion of cells to an immature or a lessdifferentiated form, and it occurs in most malignant tumors.

The term “renal cell carcinoma” and “RCC” are used interchangeablyherein, refers to a tumor of the kidney. Tumors of the kidney can bemalignant or benign and are the most common primary malignant kidneytumor. RCC usually begins in the cells that line the small tubes of eachnephron. Renal cell tumors can grow as a single mass, and can multipleRCC tumors can develop on a single kidney or both kidneys. The term RCCencompasses different subtypes of RCC, such as, but not limited toepithelial renal cell carcinoma (RCC), clear cell (conventional),papillary RCC (chromophil), chromophobe RCC, collecting duct RCC (<1%)and unclassified RCC subtypes.

The term “clear cell RCC” also referred to as “ccRCC” refers to the mostcommon renal neoplasm seen in adults (70% of tumors derived from tubularepithelium). Clear cell RCC can be as small as 1 cm or less anddiscovered incidentally, or it can be as bulky as several kilograms, andoften presents pain, as a palpable mass or with hematuria, but a widevariety of paraneoplastic syndromes have been described. Clear cell RCCmight be clinically silent for years and may present with symptoms ofmetastasis. Clear cell RCC has a characteristic gross appearance; thetumor is solid, lobulated, and yellow, with variegation due to necrosisand hemorrhage, with in some instances, the tumor circumscribed, orinvade the perirenal fat or the renal vein.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with kidney injury,e.g., AKI, chronic kidney disease or RCC. The term “treating” is notintended to cure disease or condition associated with AKI or chronickidney disease. The term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a condition, disease or disorder,e.g., a condition associated with AKI or chronic kidney disease.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers (e.g., adecrease in NPM polypeptide having a ischemic-induced phosphorylationstate), but also a cessation of, or at least slowing of, progress orworsening of symptoms compared to what would be expected in the absenceof treatment. Beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, remission (whether partial or total), and/ordecreased mortality, whether detectable or undetectable. For example,treatment is considered effective if the extent or amount of AKI orchronic kidney disease is reduced, or the progression of AKI or chronickidney disease is halted. In another example, treatment is consideredeffective if renal function is improved. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment).

As used herein, the term “treating” with respect to treatment of kidneyinjury or ischemia includes reducing or alleviating at least one adverseeffect or symptom of a condition, disease or disorder associated withkidney injury or ischemia. As used herein, the term treating is used torefer to the reduction of a symptom and/or a biochemical of kidneyinjury or ischemia by at least 10%. As a non-limiting example, atreatment can be measured by a decrease the presence of one or more of:pT86, pS88, pT95 on the NPM polypeptide and/or an increase in one ormore of pT234, pS242 of the NPM polypeptide as disclosed herein, forexample a decrease by at least 10% of the presence of one or more of:pT86, pS88, pT95 on the NPM polypeptide, or an increase by at least 10%of pT234, pS242 of the NPM polypeptide as compared to thephosphorylation status of the NPM polypeptide obtained from the subjectat an earlier timepoint. In some embodiments, the terms “treat” and“treatment” is administration of an appropriate therapy to the subjectidentified with RCC for a beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total). “Treatment”can also mean prolonging survival as compared to expected survival ifnot receiving treatment. Those in need of treatment include thosealready diagnosed with cancer as well as those likely to developsecondary tumors due to metastasis.

The term “effective amount” as used herein refers to the amount oftherapeutic agent or pharmaceutical composition to reduce or alleviateor at least one symptom or marker of the disease or disorder, forexample a symptom of ischemia, kidney injury or AKI. For example, aneffective amount using the methods as disclosed herein would beconsidered as the amount sufficient to reduce a symptom or marker of thedisease or disorder by at least 10%. An effective amount as used hereinwould also include an amount sufficient to prevent or delay thedevelopment of a symptom of the disease, alter the course of a symptomdisease (for example but not limited to, slowing the progression of asymptom of the disease), or reverse a symptom of the disease.

The term “effective amount” as used herein refers to the amount of anagent which inhibits the formation of the Bax-NPM complex, or a NPMinhibitory peptide as described herein, needed to alleviate at least oneor more symptom of the disease or disorder being treated, and relates toa sufficient amount of pharmacological composition to provide thedesired effect. The term “therapeutically effective amount” thereforerefers to an amount of an agent which inhibits the formation of theBax-NPM complex, or a NPM inhibitory peptide as described herein, usingthe methods as disclosed herein, that is sufficient to provide aparticular effect when administered to a typical subject. An effectiveamount as used herein would also include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slow the progression ofa symptom of the disease), or reverse a symptom of the disease. Thus, itis not possible to specify the exact “effective amount”. However, forany given case, an appropriate “effective amount” can be determined byone of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of an agent which inhibits the formation of the Bax-NPMcomplex, or a NPM inhibitory peptide, as described herein, whichachieves a half-maximal inhibition of measured function or activity) asdetermined in cell culture, or in an appropriate animal model. Levels inplasma can be measured, for example, by high performance liquidchromatography. The effects of any particular dosage can be monitored bya suitable bioassay. The dosage can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment.Depending on the type and severity of the chronic kidney disease, about1 μg/kg to 100 mg/kg (e.g., 0.1-20 mg/kg) of an agent which inhibits theformation of the Bax-NPM complex, or a NPM inhibitory peptide asdescribed herein is an initial candidate dosage range for administrationto the subject, whether, for example, by one or more separateadministrations, or by continuous infusion.

As used herein, the term “pharmaceutical composition” refers to the oneor more active agents in combination with a pharmaceutically acceptablecarrier e.g. a carrier commonly used in the pharmaceutical industry. Thephrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject, e.g. parenteral, intravenous,intralesional, or intratumoral. Exemplary modes of administrationinclude, but are not limited to, injection, infusion, instillation,inhalation, or ingestion. “Injection” includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In preferredembodiments, the compositions are administered by intravenous infusionor injection. The administration can be systemic or local.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutically active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce a toxic, anallergic, or similar untoward reaction when administered to a host.

The term “in vivo” refers to assays or processes that occur in or withinan organism, such as a multicellular animal. In some of the aspectsdescribed herein, a method or use can be said to occur “in vivo” when aunicellular organism, such as a bacterium, is used. The term “ex vivo”refers to methods and uses that are performed using a living cell withan intact membrane that is outside of the body of a multicellular animalor plant, e.g., explants, cultured cells, including primary cells andcell lines, transformed cell lines, and extracted tissue or cells,including blood cells, among others. The term “in vitro” refers toassays and methods that do not require the presence of a cell with anintact membrane, such as cellular extracts, and can refer to theintroducing of a programmable synthetic biological circuit in anon-cellular system, such as a medium not comprising cells or cellularsystems, such as cellular extracts.

The term “subject” as used herein refers to a human or animal, to whomtreatment, including prophylactic treatment, according to the presentinvention, is provided. Usually the animal is a vertebrate such as, butnot limited to a primate, rodent, domestic animal or game animal.Primates include but are not limited to, chimpanzees, cynomologousmonkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents includemice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and gameanimals include, but are not limited to, cows, horses, pigs, deer,bison, buffalo, feline species, e.g., domestic cat, canine species,e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, andfish, e.g., trout, catfish and salmon. In certain embodiments of theaspects described herein, the subject is a mammal, e.g., a primate or ahuman. A subject can be male or female. Additionally, a subject can bean infant or a child. In some embodiments, the subject can be a neonateor an unborn subject, e.g., the subject is in utero. Preferably, thesubject is a mammal. The mammal can be a human, non-human primate,mouse, rat, dog, cat, horse, or cow, but is not limited to theseexamples. Mammals other than humans can be advantageously used assubjects that represent animal models of diseases and disorders. Inaddition, the methods and compositions described herein can be used fordomesticated animals and/or pets. A human subject can be of any age,gender, race or ethnic group, e.g., Caucasian (white), Asian, African,black, African American, African European, Hispanic, Mideastern, etc. Insome embodiments, the subject can be a patient or other subject in aclinical setting. In some embodiments, the subject is already undergoingtreatment. In some embodiments, the subject is an embryo, a fetus,neonate, infant, child, adolescent, or adult. In some embodiments, thesubject is a human fetus, human neonate, human infant, human child,human adolescent, or human adult. In some embodiments, the subject is ananimal embryo, or non-human embryo or non-human primate embryo. In someembodiments, the subject is a human embryo.

The term “derivative” as used herein refers to proteins or peptides(e.g., NPM inhibitory peptides or fusion proteins thereof) which havebeen chemically modified, for example but not limited to by techniquessuch as ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol) or addition of other molecules.

As used herein, “variant” with reference to a polynucleotide orpolypeptide, refers to a polynucleotide or polypeptide that can vary inprimary, secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide). A “variant” of a NPMinhibitory peptides for example, is meant to refer to a moleculesubstantially similar in structure and function, i.e. where the functionis the ability to bind to biotin or a biotin derivative, or to a lipoicacid compound as disclosed herein. A molecule is said to be“substantially similar” to another molecule if both molecules havesubstantially similar structures or if both molecules possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif the structure of one of the molecules not found in the other, or ifthe sequence of amino acid residues is not identical.

For example, a variant of NPM inhibitory peptides can contain a mutationor modification that differs from a reference amino acid of SEQ ID NO: 1or SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, a variant can be adifferent isoform of a NPM protein or can comprise different isomeramino acids. Variants can be naturally-occurring, synthetic,recombinant, or chemically modified polynucleotides or polypeptidesisolated or generated using methods well known in the art. Variants caninclude conservative or nonconservative amino acid changes, as describedbelow. Polynucleotide changes can result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. Variants can also include insertions,deletions or substitutions of amino acids, including insertions andsubstitutions of amino acids and other molecules) that do not normallyoccur in the peptide sequence that is the basis of the variant, forexample but not limited to insertion of ornithine which do not normallyoccur in human proteins.

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the polypeptide's activity. For example, aconservative substitution refers to substituting an amino acid residuefor a different amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine. “Conservative amino acid substitutions” resultfrom replacing one amino acid with another having similar structuraland/or chemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine. Thus, a “conservative substitution” of a particular amino acidsequence refers to substitution of those amino acids that are notcritical for polypeptide activity or substitution of amino acids withother amino acids having similar properties (e.g., acidic, basic,positively or negatively charged, polar or non-polar, etc.) such thatthe substitution of even critical amino acids does not reduce theactivity of the peptide, (i.e. the ability of the peptide to penetratethe BBB). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984).) In some embodiments, individual substitutions, deletions oradditions that alter, add or delete a single amino acid or a smallpercentage of amino acids can also be considered “conservativesubstitutions” is the change does not reduce the activity of the peptide(i.e. the ability of, for example MIS to bind and activate MISRII).Insertions or deletions are typically in the range of about 1 to 5 aminoacids. The choice of conservative amino acids may be selected based onthe location of the amino acid to be substituted in the peptide, forexample if the amino acid is on the exterior of the peptide and exposeto solvents, or on the interior and not exposed to solvents. As usedherein, the term “nonconservative” refers to substituting an amino acidresidue for a different amino acid residue that has different chemicalproperties. The nonconservative substitutions include, but are notlimited to aspartic acid (D) being replaced with glycine (G); asparagine(N) being replaced with lysine (K); or alanine (A) being replaced witharginine (R).

The terms “insertion” or “deletion” are typically in the range of about1 to 5 amino acids. The variation allowed can be experimentallydetermined by producing the peptide synthetically while systematicallymaking insertions, deletions, or substitutions of nucleotides in thesequence using recombinant DNA techniques.

The term “functional derivative” and “mimetic” are used interchangeably,and refers to a compound which possess a biological activity (eitherfunctional or structural) that is substantially similar to a biologicalactivity of the entity or molecule its is a functional derivative of.The term functional derivative is intended to include the fragments,variants, analogues or chemical derivatives of a molecule.

A “fragment” of a molecule, is meant to refer to any contagiouspolypeptide subset of the molecule. Fragments of, for example a NPMpolypeptide of SEQ ID NO: 4 which have the same activity as that ofamino acid of SEQ ID NO: 2 or SEQ ID N: 3 are also encompassed for usein the present invention.

An “analog” of a molecule such as a NPM inhibitory peptide, for examplean analogue of the protein of amino acid of SEQ ID NO: 1 or SEQ ID NO: 2or SEQ ID NO: 3 is meant to refer to a molecule similar in function toeither the entire molecule or to a fragment thereof of SEQ ID NO: 1 orSEQ ID NO: 2 or SEQ ID NO: 3, respectively. As used herein, a moleculeis said to be a “chemical derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half life, etc. The moieties can alternativelydecrease the toxicity of the molecule, eliminate or attenuate anyundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, Pa.(1990).

As used herein, “homologous”, when used to describe a polypeptide orpolynucleotide, indicates that two polypeptides or two polynucleotides,or designated sequences thereof, when optimally aligned and compared,are identical, with appropriate amino acid or nucleotide insertions ordeletions, in at least 70% of the amino acids or nucleotides, usuallyfrom about 75% to 99%, and more preferably at least about 98 to 99% ofthe amino acids or nucleotides.

The term “homolog” or “homologous” can also be used with respect tostructure and/or function. With respect to amino acid sequence homology,amino acid sequences are homologs if they are at least 50%, at least 60at least 70%, at least 80%, at least 90%, at least 95% identical, atleast 97% identical, or at least 99% identical. The term “substantiallyhomologous” refers to sequences that are at least 90%, at least 95%identical, at least 97% identical or at least 99% identical. Homologoussequences can be the same functional gene in different species.

As used herein, the term “substantial similarity” in the context ofpolypeptide sequences, indicates that the polypeptide comprises asequence with at least 60% sequence identity to a reference sequence, or70%, or 80%, 85% or 87% sequence identity to the reference sequence, ormost preferably 90% identity over a comparison window of about 10-20amino acid residues. In some embodiments, a NPM inhibitory peptide withsubstantial similarity to SEQ ID NO: 1 is a peptide that has at leastabout 70%, or about 80%, or about 85% or about 87% or about 90% or moresequence identity to SEQ ID NO: 1, and can have a similar biologicalfunction or activity, e.g., at least 80% ability to inhibit the Bax-NPMcomplex formation as compared to the inhibitory NPM peptide of SEQ IDNO: 1.

In the context of amino acid sequences, “substantial similarity” furtherincludes conservative substitutions of amino acids. Thus, a polypeptideis substantially similar to a second polypeptide, for example, where thetwo peptides differ by one or more conservative substitutions. The term“substantial identity” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan. The terms“homology” or “identity” or “similarity” are used interchangeably hereinand refers to sequence similarity between two peptides or between twonucleic acid molecules. Homology and identity can each be determined bycomparing a position in each sequence which can be aligned for purposesof comparison. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar amino acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, though preferably less than 25% identity with asequence of the present application.

In one embodiment, the term “NPM inhibitory peptide homolog” refers toan amino acid sequence that has 40% homology to the reference sequenceof the NPM inhibitory peptide. Using SEQ ID NO: 1 as the exemplary NPMinhibitory peptide as disclosed herein, a NPM inhibitory peptide homologhas 40% sequence identity or more preferably at least about 50%, stillmore preferably, at least about 60% sequence identity, still morepreferably, at least about 70% sequence identity, even more preferably,at least about 75% sequence identity, yet more preferably, at leastabout 80% sequence identity, even more preferably at least about 85%homology, still more preferably, at least about 90% sequence identity,and more preferably, at least about 95% sequence identity to the aminoacids of SEQ ID NO: 1. As discussed above, homology refers to a sequenceidentity of between about 40% to 100% and all intervals in between(i.e., 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.).

The term “sequence identity” with reference to nucleic acid sequencesrefers to the relatedness between two nucleotide sequences. For purposesof the present disclosure, the degree of sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0 or later. The optional parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSSversion of NCBI NUC4.4) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows: (IdenticalDeoxyribonucleotides.times.100)/(Length of Alignment-Total Number ofGaps in Alignment). The length of the alignment is preferably at least10 nucleotides, preferably at least 25 nucleotides more preferred atleast 50 nucleotides and most preferred at least 100 nucleotides.

The term “homology” or “homologous” as used herein is defined as thepercentage of nucleotide residues in the homology arm that are identicalto the nucleotide residues in the corresponding sequence on the targetchromosome, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleotide sequence homology can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. Those skilledin the art can determine appropriate parameters for aligning sequences,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. In some embodiments, anucleic acid sequence (e.g., DNA sequence), for example of a homologyarm of a repair template, is considered “homologous” when the sequenceis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or more, identicalto the corresponding native or unedited nucleic acid sequence (e.g.,genomic sequence) of the host cell.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level or non-detectable level as compared to a referencesample), or any decrease between 10-100% as compared to a referencelevel. In the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers to astandard definition of statistical significance and generally means atwo standard deviation (2SD) below normal, or lower, concentration ofthe marker. The term refers to statistical evidence that there is adifference. It is defined as the probability of making a decision toreject the null hypothesis when the null hypothesis is actually true.The decision is often made using the p-value.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment. The use of “comprising”indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%. The present invention is further explained in detail by thefollowing examples, but the scope of the invention should not be limitedthereto.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Unless otherwisestated, the present invention was performed using standard procedures,as described, for example in Sambrook et al., Molecular Cloning: ALaboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2001); Davis et al., Basic Methods inMolecular Biology, Elsevier Science Publishing, Inc., New York, USA(1995); Current Protocols in Protein Science (CPPS) (John E. Coligan,et. al., ed., John Wiley and Sons, Inc.), Current Protocols in CellBiology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons,Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R.Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal CellCulture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather andDavid Barnes editors, Academic Press, 1st edition, 1998) which are allincorporated by reference herein in their entireties. Other terms aredefined herein within the description of the various aspects of theinvention.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

II. NPM

Nucleophosmin (NPM), also known as B23, NQ38, or numatrin (Lim and Wang,2006), is a nucleolar phosphoprotein composed of an N-terminal globulardomain (1-110 residues) and a C-terminal domain (111-294 residues) richin acidic residues. NPM was initially identified as a critical player inribosome biogenesis (Lim and Wang, 2006). Since then a number ofcellular activities associated with NPM indicate that this protein hasmultiple functions, especially in ceil proliferation,cytoplasmic/nuclear shuttle transportation, nucleic acid binding,ribonucleic cleavage, centrosome duplication and molecular chaperoning(Okuda, 2002; Okuwaki et al, 2001; Ye, 2005), NPM shuttles between thenucleolus and the cytoplasm, and it also translocates from the nucleolusto the nucleoplasm during the stationary phase of growth or duringtreatment with certain antitumor drugs (Chou and Yung, 1995; Yung et al,1990).

Nucleophosmin is also known as, NPM, NPM1, nucleophosmin 1, B23,nucleolar phosphoprotein B23, numatrin, nucleophosmin/nucleoplasminfamily, member 1. The sequence of NPM for a number of species is wellknown in the art, e.g. human NPM (e.g. SEQ ID NO: 4, NCBI Ref Seq:NP_002511.1), and is encoded by NM_002520.6. The amino acid sequence ofhuman NPM polypeptide is as follows, with the 5 serine (S) or threonine(T) sites (T86, S88, T95, T234 or S242) that are differentiallyphosphorylated under ischemic condition highlighted in bold and italics:

(SEQ ID NO: 4) medsmdmdms plrpqnylfg celkadkdyh fkvdndenehqlslrtvslg agakdelhiv eaeamnyegs pikvtlatlk msvqp t v s lg gfei tppvvl rlkcgsgpvh isgqhlvave edaesedeee edvkllsisg krsapgggsk vpqkkvklaadeddddddee dddedddddd fddeeaeeka pvkksirdtpaknaqksnqn gkdskpsstp rskgqesfkk qek t pktpkg p ssvedikak mgasiekggs lpkveakfin yvkncfrmtd geaiqdlwqw rksl

The NPM polypeptide of SEQ ID NO: 4 is encoded by RefSeq ID:NM_002520.6, which is as follows:

(SEQ ID NO: 7) 1 agaaaggagt ggggttgaaa agcgcttgcg caggacggctacggtacggg ggtgggaggg 61 cttcggagca cgcgcgcgga ggcgggactt gggaagcgctcgcgagatct tcagggtcta 121 tatataagcg cggggagcct gcgtcctttc cctggtgtgattccgtcctg cgcggttgtt 181 ctctggagca gcgttctttt atctccgtcc gccttctctcctacctaagt gcgtgccgcc 241 acccgatgga agattcgatg gacatggaca tgagccccctgaggccccag aactatcttt 301 tcggttgtga actaaaggcc gacaaagatt atcactttaaggtggataat gatgaaaatg 361 agcaccagtt atctttaaga acggtcagtt taggggctggtgcaaaggat gagttgcaca 421 ttgttgaagc agaggcaatg aattacgaag gcagtccaattaaagtaaca ctggcaactt 481 tgaaaatgtc tgtacagcca acggtttccc ttgggggctttgaaataaca ccaccagtgg 541 tcttaaggtt gaagtgtggt tcagggccag tgcatattagtggacagcac ttagtagctg 601 tggaggaaga tgcagagtca gaagatgaag aggaggaggatgtgaaactc ttaagtatat 661 ctggaaagcg gtctgcccct ggaggtggta gcaaggttccacagaaaaaa gtaaaacttg 721 ctgctgatga agatgatgac gatgatgatg aagaggatgatgatgaagat gatgatgatg 781 atgattttga tgatgaggaa gctgaagaaa aagcgccagtgaagaaatct atacgagata 841 ctccagccaa aaatgcacaa aagtcaaatc agaatggaaaagactcaaaa ccatcatcaa 901 caccaagatc aaaaggacaa gaatccttca agaaacaggaaaaaactcct aaaacaccaa 961 aaggacctag ttctgtagaa gacattaaag caaaaatgcaagcaagtata gaaaaaggtg 1021 gttctcttcc caaagtggaa gccaaattca tcaattatgtgaagaattgc ttccggatga 1081 ctgaccaaga ggctattcaa gatctctggc agtggaggaagtctctttaa gaaaatagtt 1141 taaacaattt gttaaaaaat tttccgtctt atttcatttctgtaacagtt gatatctggc 1201 tgtccttttt ataatgcaga gtgagaactt tccctaccgtgtttgataaa tgttgtccag 1261 gttctattgc caagaatgtg ttgtccaaaa tgcctgtttagtttttaaag atggaactcc 1381 accctttgct tggttttaag tatgtatgga atgttatgataggacatagt agtagcggtg 1381 gtcagacatg gaaatggtgg ggagacaaaa atatacatgtgaaataaaac tcagtatttt 1441 aataaagta

The inventors have previously discovered that nucleophosmin (NPM), ahighly conserved, ubiquitously expressed nucleolar protein essential formammalian cell survival, 16-18 also facilitates PTEC death in aBax-dependent manner.13 In normal cells, NPM acts as a protein chaperonethat shuttles between the nucleus and cytosol to promote proteinsynthesis, ribosomal biogenesis, and cell proliferation.13,17,19,20,During ischemic stress, however, NPM rapidly enters the cytosol andcomplexes with conformationally activated Bax, and together, NPM and Baxcause mitochondrial injury and cell death.13 NPM exists in two forms:large multimers which are restricted to the nucleus and monomers capableof entering the cytosol.21 In general, oligomeric NPM promotes cellproliferation, whereas monomeric NPM enhances death21 after proapoptoticinsults that activate Bax in both cancer22,23 and noncancer13 cells. Thepost-translational modifications that convert NPM from its essentialrole in normal cell housekeeping to a cytotoxin during stress arecompletely unknown.

Interestingly, neither conformational Bax activation or cytosolic NPMaccumulation alone are toxic, suggesting that both events are requiredto induce cell death¹³. Prior experimental evidence shows that NPMde-oligomerizes to form monomers, translocates from the nucleus to thecytosol in order to bind Bax in its conformationally altered, i.e.,“active” form that exposes the 6A7 epitope^(22,23,24,25).

In particular, the authors investigated stress-induced differentialphosphorylation of nucleophosmin (NPM), a chaperone of the apoptoticfactor Bax, finding a virtually identical NPM stress-inducedphosphorylation pattern in mouse and human primary renal cells, freshkidney tissue, and urine within hours of injury. An NPM mimic proteinthat replicates this pattern (but not a mimic with a normal pattern) istoxic to renal cells. Administering targeted peptides designed to reduceNPM toxicity, even hours after a typically lethal ischemic insult,improves cell and animal survival. As such, the inventors demonstratethat stress-induced NPM phosphorylation is a contributor to renal celldeath in human AKI and specific ischemic-induced NPM phosphorylationchanges that potentially guide early diagnosis and management

III. NPM Inhibitor Agents and Inhibitors of Bax-NPM Complex Formation asTherapeutics for AKI and Renal Injury

One aspect of the technology described herein relates to a method totreat a subject any of: acute kidney injury (AKI) or ischemia,comprising administering a subject a composition comprising at least oneagent which inhibits the formation of the Bax-NPM complex. In someembodiments, an inhibitor of a Bax-NPM complex is a NPM inhibitorypeptide as discussed herein. In some embodiments, a NPM inhibitorypeptide selected from the group consisting of: TVTIFVAGVLTASLTIWKKMG(SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (peptide #2) (SEQ ID NO: 2)and ESFKKQEKTPKTPKGPSSVEDIKAK (peptide #3) (SEQ ID NO: 3), or a peptidewith at least 85% or 90% or 95% sequence identity to any of SEQ ID NO:1-3.

As used herein, an agent which inhibits the formation of the Bax-NPMcomplex, or a NPM inhibitory peptide as disclosed herein has the abilityto reduce the activity of the ischemic-induced phosphorylated form ofthe NPM polypeptide (i.e., having the phosphorylation status of one ormore of pT86, pS88, pT95, nT234, nS242) and/or its binding to Bax (i.e.,inhibit Bax-NPM complex formation) by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or more, relativeto the activity of the normal form of the NPM polypeptide (i.e., havingthe phosphorylation status of nT86, nS88, nT95, pT234, pS242) or bindingof the normal NPM polypeptide to Bax level in the absence of the NPMpeptide.

As used herein, the terms an “agent which inhibits the formation of theBax-NPM complex”, “Bax-NPM complex inhibitor”, “NPM antagonist,” “NPMinhibitor peptide,” and “NPM inhibitor agent” refer to a molecule oragent that significantly blocks, inhibits, reduces, or interferes withthe ischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242) biological activity in vitro, in situ, and/or in vivo,including activity of downstream signalling pathways mediated by thephosphorylated form of the NPM polypeptide, such as, for example, NPMinteraction with Bax and/or complex formation with Bax to form a Bax-NPMcomplex, and down stream Bax-mediated cell death, and/or elicitation ofa cellular response to Bax or the Bax-NPM complex,

The term “agent” as used herein in reference to an agent which inhibitsthe formation of the Bax-NPM complex, or a NPM inhibitor agent means anycompound or substance such as, but not limited to, a small molecule,nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can beany chemical, entity, or moiety, including, without limitation,synthetic and naturally-occurring proteinaceous and non-proteinaceousentities. In some embodiments of the aspects described herein, an agentis a nucleic acid, a nucleic acid analogue, a protein, an antibody, apeptide, an aptamer, an oligomer of nucleic acids, an amino acid, or acarbohydrate, and includes, without limitation, proteins,oligonucleotides, ribozymes, DNAzymes, glycoproteins, antisense RNAs,siRNAs, lipoproteins, aptamers, and modifications and combinationsthereof etc. Compounds for use in the therapeutic compositions andmethods described herein can be known to have a desired activity and/orproperty, or can be selected from a library of diverse compounds, usingscreening methods known to one of ordinary skill in the art,

Exemplary agents which inhibits the formation of the Bax-NPM complex, ora NPM inhibitor agent contemplated for use in the various aspects andembodiments described herein include, but. are not limited to, anti-NPMantibodies or antigen-binding fragments thereof that specifically bindto the ischemic-induced phosphorylated form of the NPM polypeptide(i.e., having the phosphorylation status of one or more of pT86, pS88,pT95, nT234, nS242); anti-sense molecules directed to a nucleic acidencoding NPM polypeptide; short interfering RNA (“siRNA”) moleculesdirected to a nucleic acid encoding NPM polypeptide; RNA or DNA aptamersthat bind to the ischemic-induced phosphorylated form of the NPMpolypeptide (i.e., having the phosphorylation status of one or more ofpT86, pS88, pT95, nT234, nS242), and inhibit/reduce/block signaling bythe ischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242), structural analogs of the ischemic-induced phosphorylatedform of the NPM polypeptide (i.e., having the phosphorylation status ofone or more of pT86, pS88, pT95, nT234, nS242); and soluble proteins ofthe ischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242), inhibitory NPM polypeptides, e.g., NPM inhibitorypeptides as disclosed herein, e.g., dominant negative NPM inhibitorypeptides, or fusion polypeptides thereof. In some embodiments of theseaspects and all such aspects described herein, an inhibitor of theformation of the Bax-NPM complex, or a NPM inhibitor agent (e.g., anantibody or antigen-binding fragment thereof) binds (physicallyinteracts with) the ischemic-induced phosphorylated form of the NPMpolypeptide (i.e., having the phosphorylation status of one or more ofpT86, pS88, pT95, nT234, nS242), targets downstream signaling of theischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242), and/or inhibits (reduces) the change of phosphorylationstates of the NPM polypeptide from the normal phosphorylation state(e.g., nT86, nS88, nT95, pT234, pS242) to the ischemic-inducedphosphorylated form having the phosphorylation status of one or more ofpT86, pS88, pT95, nT234, nS242), as well as NPM polypeptide productionor release. In some embodiments of these aspects and all such aspectsdescribed herein, an inhibitor of the formation of the Bax-NPM complex,or a NPM inhibitor agent binds and prevents its binding Bax. In someembodiments of these aspects and ail such aspects described herein, aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent specifically reduces or eliminates cell death mediated by theBax-NPM complex,

As used herein, an inhibitor of the formation of the Bax-NPM complex, ora NPM inhibitor agent has the ability to reduce the formation of theBax-NPM complex in a ceil (e.g., in a ischemic cell, e.g., a podocytes)by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or more, relative to the amount of Bax-NPM complex formed inthe absence of the an inhibitor of the formation of the Bax-NPM complex,or a NPM inhibitor agent.

In some embodiments of the compositions and methods described herein, anagent which inhibits the formation of the Bax-NPM complex is a smallmolecule compound or agent that targets or binds to ischemic-inducedphosphorylated form of the NPM polypeptide (i.e., having thephosphorylation status of one or more of pT86, pS88, pT95, nT234,nS242), including, but not limited to, small peptides or peptide-likemolecules, soluble peptides, and synthetic non-peptidyl organic orinorganic compounds. As used herein, the term “small molecule” refers toa chemical agent which can include, but is not limited to, a peptide, apeptidomimetic, an amino acid, an amino acid analog, a polynucleotide, apolynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, anorganic or inorganic compound (e.g., including heterorganic andorganometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. Exemplary sites of small moleculebinding include, but are not limited to, the portion of NPM polypeptidethat binds to Bax, or portions of the NPM polypeptide comprising any oneor more of T86, S88, T95, T234, S242, or bind to at least 5 amino acidsanywhere within amino acids 78-103 of SEQ ID NO: 4 or bind to at least 5amino acids anywhere within amino acids 226-246 of SEQ ID NO: 4.

In some embodiments of the compositions and methods described herein, aninhibitor of Bax-NPM complex formation comprises a small molecule thatbinds to the ischemic-induced phosphorylated form of the NPM polypeptide(i.e., having the phosphorylation status of one or more of pT86, pS88,pT95, nT234, nS242) and inhibits its biological activity.

In some embodiments of the compositions and methods described herein,the binding sites of an inhibitor of the formation of the Bax-NPMcomplex, or a NPM inhibitor agent, such as an antibody orantigen-binding fragment thereof, are directed against a NPM interactionsite, such as its binding site with Bax. In some embodiments of thecompositions and methods described herein, the binding sites of aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent are directed against at least one of the stress-inducedphosphorylation sites, e.g., any one or more of T86, S88, T95, T234,S242 on the NPM polypeptide of SEQ ID NO: 4, or a site in closeproximity of these phosphorylation sites, in order to provide sterichindrance for the interaction of kinases, and/or phosphatases, orinteraction with Bax. By binding to a Bax binding site, or to a regionon the NPM polypeptide of SEQ ID NO: 4 that is confirmationally locatedclose to one or more sites of T86, S88, T95, T234, S242 on SEQ ID NO: 4,an inhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent described herein can reduce or inhibit the binding of theischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242) to Bax, and downstream signaling consequences of theBax-NPM complex. For example, in some embodiments of the compositionsand methods described herein, the binding sites of an inhibitor of theformation of the Bax-NPM complex, or a NPM inhibitor agent block ortarget amino acids 78-103 of SEQ ID NO: 4 or amino acids 226-246 of SEQID NO: 4, and preferably both amino acids 78-103 of SEQ ID NO: 4 oramino acids 226-246 of SEQ ID NO: 4, i.e., NPM(78-103) and/orNPM(226-246) respectively of SEQ ID NO: 4, for example. This can beaccomplished by a variety of means well known in the art, such asantibodies and antigen-binding fragments thereof, inhibitor RNAs, etc.,and as described herein.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, an inhibitor of the formation of the Bax-NPM complex,or a NPM inhibitor agent is an antibody or antigen-binding fragmentthereof that selectively binds or physically interacts with theischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242). In some embodiments of the compositions and methodsdescribed herein, an inhibitor of the formation of the Bax-NPM complex,or a NPM inhibitor agent is an antibody or antigen-binding fragmentthereof that binds to the oligomeric form of the NPM polypeptide andinhibits and/or blocks and/or prevents interaction with Bax or Bax-NPMcomplex formation. In some embodiments of the compositions and methodsdescribed herein, the antibody or antigen-binding fragment thereof bindsto the ischemic-induced phosphorylated form of the NPM polypeptide(i.e., having the phosphorylation status of one or more of pT86, pS88,pT95, nT234, nS242). In some embodiments of the compositions and methodsdescribed herein, the antibody or antigen-binding fragment thereof bindsto the ischemic-induced phosphorylated form of the NPM polypeptide(i.e., having the phosphorylation status of one or more of pT86, pS88,pT95, nT234, nS242) can bind to block or target amino acids 78-103 ofSEQ ID NO: 4 or amino acids 226-246 of SEQ ID NO: 4, and preferably bothamino acids 78-103 of SEQ ID NO: 4 or amino acids 226-246 of SEQ ID NO:4, i.e., NPM(78-103) and/or NPM(226-246) respectively of SEQ ID NO: 4.In some embodiments of the compositions and methods described herein,the antibody or antigen-binding fragment thereof binds to or blocks theBax-NPM interaction. In some embodiments of the compositions and methodsdescribed herein, the antibody or antigen-binding fragment thereof bindsto or blocks the Bax-binding site of NPM polypeptide.

Antibodies specific for or that selectively bind the ischemic-inducedphosphorylated form of the NPM polypeptide (i.e., having thephosphorylation status of one or more of pT86, pS88, pT95, nT234, nS242)suitable for use in the compositions and for practicing the methodsdescribed herein are preferably monoclonal, and can include, but are notlimited to, human, humanized or chimeric antibodies, comprising singlechain antibodies, Fab fragments, F(ab′) fragments, fragments produced bya Fab expression library, and/or binding fragments of any of the above.Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain antigen or target binding sites or “antigen-binding fragments.”The immunoglobulin molecules described herein can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass of immunoglobulin molecule, as is understoodby one of skill in the art.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, an inhibitor of the formation of the Bax-NPM complex,or a NPM inhibitor agent as described herein is a monoclonal anti-NPMantibody or antigen-binding fragment.

In some embodiments of the compositions and methods described herein, aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent as described herein is an anti-NPM antibody fragment orantigen-binding fragment. In some embodiments, the anti-NPM antibodyspecifically binds to the ischemic-induced phosphorylated form of theNPM polypeptide (i.e., having the phosphorylation status of one or moreof pT86, pS88, pT95, nT234, nS242) and does not bind to the normal NPMpolypeptide (having a phosphorylation status of nT86, nS88, nT95, pT234,pS242). The terms “antibody fragment,” “antigen binding fragment,” and“antibody derivative” as used herein, refer to a protein fragment thatcomprises only a portion of an intact antibody, generally including anantigen binding site of the intact antibody and thus retaining theability to bind antigen. Examples of antibody fragments encompassed bythe terms antibody fragment or antigen-binding fragment include: (i) theFab fragment, having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) theFab′ fragment, which is a Fab fragment having one or more cysteineresidues at the C-terminus of the C_(H)1 domain; (iii) the Fd fragmenthaving V_(H) and C_(H)1 domains; (iv) the Fd′ fragment having V_(H) andC_(H)1 domains and one or more cysteine residues at the C-terminus ofthe C_(H)1 domain; (v) the Fv fragment having the V_(L) and V_(H)domains of a single arm of an antibody; (vi) a dAb fragment (Ward etal., Nature 341, 544-546 (1989)) which consists of a V_(H) domain or aV_(L) domain; (vii) isolated CDR regions; (viii) F(ab′)₂ fragments, abivalent fragment including two Fab′ fragments linked by a disulphidebridge at the hinge region; (ix) single chain antibody molecules (e.g.single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); andHuston et al., PNAS (USA) 85:5879-5883 (1988)); (x) “diabodies” with twoantigen binding sites, comprising a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) “linearantibodies” comprising a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions (Zapata etal. Protein Eng. 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870);and modified versions of any of the foregoing (e.g., modified by thecovalent attachment of polyalkylene glycol (e.g., polyethylene glycol,polypropylene glycol, polybutylene glycol) or other suitable polymer).

In some embodiments of the compositions and methods described herein, aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent or antagonist is a chimeric antibody derivative of a an antibodythat specifically binds to the ischemic-induced phosphorylated form ofthe NPM polypeptide (i.e., having the phosphorylation status of one ormore of pT86, pS88, pT95, nT234, nS242) and does not bind to the normalNPM polypeptide (having a phosphorylation status of nT86, nS88, nT95,pT234, pS242) or antigen-binding fragment thereof. In some embodiments,an inhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent or antagonist antibodies and antigen-binding fragments thereofdescribed herein can also be, in some embodiments, a humanized antibodyderivative.

In some embodiments, an inhibitor of the formation of the Bax-NPMcomplex, or a NPM inhibitor agent or antagonist antibodies andantigen-binding fragments thereof described herein include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the antibody, provided that the covalent attachment does notprevent the antibody from binding to the target antigen, e.g., theischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242).

In some embodiments of the compositions and methods described herein,completely human antibodies are used, which are particularly desirablefor the therapeutic treatment of human patients.

In some embodiments of the compositions and methods described herein, aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent comprises at least one antisense molecule capable of blocking ordecreasing the expression of a particular functional NPM polypeptide bytargeting nucleic acids encoding NPM, or relevant domains thereof. Insome embodiments of the compositions and methods described herein, theat least one antisense molecule targets nucleic acids encoding the Baxbinding domain of the ischemic-induced phosphorylated form of the NPMpolypeptide (i.e., having the phosphorylation status of one or more ofpT86, pS88, pT95, nT234, nS242). In some embodiments of the compositionsand methods described herein, the at least one antisense moleculetargets nucleic acids encoding the Bax binding domain. Methods are knownto those of ordinary skill in the art for the preparation of antisenseoligonucleotide molecules that will specifically bind NPM mRNA withoutcross-reacting with other polynucleotides. Exemplary sites of targetinginclude, but are not limited to, the initiation codon, the 5′ regulatoryregions, including promoters or enhancers, the coding sequence,including any conserved consensus regions, and the 3′ untranslatedregion. In some embodiment of these aspects and all such aspectsdescribed herein, the antisense oligonucleotides are about 10 to about100 nucleotides in length, about 15 to about 50 nucleotides in length,about 18 to about 25 nucleotides in length, or more. In certainembodiments, the antisense oligonucleotides further comprise chemicalmodifications to increase nuclease resistance and the like, such as, forexample, phosphorothioate linkages and 2′-O-sugar modifications known tothose of ordinary skill in the art.

In some embodiments of the compositions and methods described herein, aninhibitor of the formation of the Bax-NPM complex, or a NPM inhibitoragent comprises at least one short interfering RNA (siRNA) moleculecapable of blocking or decreasing the expression of functional NPM bytargeting nucleic acids encoding SEQ ID NO: 4 (e.g., SEQ ID NO: 7), orrelevant domains thereof. In some embodiments of the compositions andmethods described herein, the at least one siRNA molecule targetsnucleic acids encoding the Bax binding domain of NPM.

In some embodiments of the compositions and methods described herein,the at least one siRNA molecule targets nucleic acids encoding the Baxbinding domain of NPM polypeptide. In some embodiments of thecompositions and methods described herein, the at least one siRNAmolecule targets nucleic acids encoding the regions of the NPMpolypeptide comprising at least one or more of amino acids T86, S88,T95, T234, S242. It is routine to prepare siRNA molecules that willspecifically target NPM mRNA without cross-reacting with otherpolynucleotides. siRNA molecules for use in the compositions and methodsdescribed herein can be generated by methods known in the art, such asby typical solid phase oligonucleotide synthesis, and often willincorporate chemical modifications to increase half-life and/or efficacyof the siRNA agent, and/or to allow for a more robust deliveryformulation. Alternatively, siRNA molecules are delivered using a vectorencoding an expression cassette for intracellular transcription ofsiRNA.

In some embodiments of the compositions and methods described herein, aRNA or DNA aptamer binds to or physically interacts with theischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242), i.e., NPM (78-103) of SEQ ID NO: 4 or NPM(226-246) of SEQID NO: 4. In some embodiments of the compositions and methods describedherein, the RNA or DNA aptamer binds to or physically interacts with orblocks the ischemic-induced phosphorylated form of the NPM polypeptidefrom binding to Bax.

In some embodiments of the compositions and methods described herein, aninhibitor of Bax-NPM complex formation comprises at least one NPMstructural analog, such as a dominant negative NPM polypeptide. In someembodiments of the compositions and methods described herein, aninhibitor of Bax-NPM complex formation comprises at least one solubleNPM peptide or fusion polypeptide thereof, such as, for example, a NPMinhibitory polypeptide. In some such embodiments, the NPM inhibitorypolypeptide is a dominant negative NPM fusion protein. In someembodiments of the compositions and methods described herein, the NPMinhibitory polypeptide comprises amino acids 78-103 of SEQ ID NO: 4 oramino acids 226-246 of SEQ ID NO: 4 of variants having at least 50%, or60%, or at least 70%, or at least 80%, or at least 90% or at least 95%or at least 98% sequence identity thereto.

An inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein, for use in the compositions and methods describedherein can be identified or characterized using methods known in theart, such as protein-protein binding assays, biochemical screeningassays, immunoassays, and cell-based assays, which are well known in theart, including, but not limited to, those described herein in theExamples.

Such identified inhibitors of Bax-NPM complex formation or a NPM peptideinhibitors s can further be tested using in vivo animal models ofchronic kidney disease, such as glomerular and interstitial injurymodels (e.g., animal models of lupus nephritis, including mice of theNZB, (NZB×NZW) F1 hybrid (termed NZB/W), and congenic derivativesthereof, MRL/lpr and BXSB strains), animal models of aging (e.g., agedSprague Dawley rats and aged C57BL/6 mice); spontaneously hypertensiverats (SHR); Buffalo/mna rats, which are a model of human idiopathicnephrotic syndrome; Munich Wistar Fromter (MWF) rats, which are agenetic model related to a congenital deficit in nephron number beingpredisposed to the development of hypertension and salt sensitivity inadulthood; primary podocyte-specific genetic FSGS models; HIV-associatednephropathy (HIVAN) transgenic mouse models; animal models of Alportsyndrome, which comprise mutations of the α3, α4, or α5 chains of typeIV collagen (COL4A3, COL4A4, and COL4A5); immune-induced models, such asthe Thy-1 nephritis model, which is an experimental rat model ofmesangioproliferative glomerulonephritis (MsPGN), anti-glomerularbasement membrane (GBM) models; and non-immune induced models.

One aspect of the technology described herein relates to a method totreat a subject any of: acute kidney injury (AKI) or ischemia,comprising administering a subject a composition comprising at least onepeptide selected from the group consisting of: TVTIFVAGVLTASLTIWKKMG(SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (peptide #2) (SEQ ID NO: 2)and ESFKKQEKTPKTPKGPSSVEDIKAK (peptide #3) (SEQ ID NO: 3), or a peptidewith at least 85% or 90% or 95% sequence identity to any of SEQ ID NO:1-3. In some embodiments, the peptides of SEQ ID NO: 1-3 are conjugatedor attached to renal targeting moieties or peptides, including renaltargeting nuclear localization sequences (NLS), so the peptides targetthe kidney.

Accordingly, in some aspects, provided herein are methods for thetreatment of chronic kidney disease in a subject in need thereof, suchmethod comprising administering to a subject having or at risk for akidney disease, including chronic kidney disease a therapeuticallyeffective amount of a composition comprising a NPM peptide.

Also provided herein, in some aspects, are methods for the reduction ofproteinuria in a subject in need thereof, comprising administering to asubject having or at risk for proteinuria a therapeutically effectiveamount of a composition comprising a NPM peptide. In other aspects,provided herein are methods for preventing kidney diseases or promotingprophylaxis of kidney diseases in a subject in need thereof, comprisingadministering to a subject a therapeutically effective amount of acomposition comprising a NPM peptide so as to prevent or promoteprophylaxis of kidney disease in the subject.

Also provided herein, in some aspects, are methods for mitigating theeffects of kidney disease, reducing the severity of kidney disease,reducing the likelihood of developing kidney disease and/or slowing theprogression of kidney disease in a subject in need thereof.

Accordingly, in some embodiments of the methods described herein, aneffective amount of a composition comprising an inhibitor of Bax-NPMcomplex formation or a NPM peptide inhibitor, as described herein isadministered to a subject in order to alleviate a symptom of chronickidney disease. As used herein, “alleviating a symptom chronic kidneydisease” is ameliorating any condition or symptom associated with thechronic kidney disease. Alternatively, alleviating a symptom of achronic kidney disease can involve reducing one or more symptoms of thechronic kidney disease in the subject relative to an untreated controlsuffering from chronic kidney disease or relative to the subject priorto the treatment. As compared with an equivalent untreated control, orthe subject prior to the treatment with an inhibitor of Bax-NPM complexformation or a NPM peptide inhibitor, as described herein, suchreduction or degree of prevention is at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more, as measured by any standard technique.Desirably, the chronic kidney disease is significantly reduced orundetectable, as detected by any standard method known in the art, inwhich case the chronic kidney disease is considered to have beentreated. A patient who is being treated for a chronic kidney disease isone who a medical practitioner has diagnosed as having such a condition.Diagnosis can be by any suitable means known to one of ordinary skill inthe art. Diagnosis and monitoring can involve, for example, detectingthe level of specific proteins or molecules in a urine, blood, or serumsample, such as, for example, albumin, calcium, cholesterol, completeblood count (CBC), electrolytes, magnesium, phosphorous, potassium,sodium, or any combination thereof; assays to detect, for example,creatinine clearance; creatinine levels; BUN (blood urea nitrogen);through the use of specific techniques or procedures, such as anabdominal CT scan, abdominal MRI, abdominal ultrasound, kidney biopsy,kidney scan, kidney ultrasound; via detection of changes in results ofassays or tests for erythropoietin, PTH; bone density test, or VitaminD; or any combination of such detection methods and assays.

A. NPM Peptide Inhibitors for Treatment of Kidney Injury, AKT andIschemia

Accordingly, one aspect of the technology described herein relates to amethod to treat a subject any of: acute kidney injury (AKI) or ischemia,comprising administering a subject a composition comprising an agentthat blocks Bax from interacting with the ischemic-inducedphosphorylated form of the NPM polypeptide (i.e., having thephosphorylation status of one or more of pT86, pS88, pT95, nT234, nS242.

In some embodiments, the agent is a NPM inhibitory peptide, e.g., apeptide or fragment from SEQ ID NO: 4. In some embodiments, a NPMinhibitory peptide does not inhibit the oligomeric form of NPMpolypeptide. In some embodiments, a NPM inhibitory peptide specificallyinhibits the monomeric form of NPM polypeptide that binds or complexeswith Bax.

In some embodiments, a NPM peptide is selected from a peptide comprisingany of: TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK(peptide #2) (SEQ ID NO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK (peptide #3)(SEQ ID NO: 3), or a peptide with at least 85% or 90% or 95% sequenceidentity to any of SEQ ID NO: 1-3.

In some embodiments, a NPM inhibitory peptide comprises an amino acidsequence having at least 50% amino acid identity, at least 55% identity,at least 60% identity, at least 65% identity, at least 70% identity, atleast 75% identity, at least 80% identity, preferably at least 85%identity, at least 90% identity, at least 95% amino acid identity, atleast 96% amino acid identity, at least 97% amino acid identity, atleast 98% amino acid identity, or at least 99% amino acid sequenceidentity to any of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, a NPM inhibitory peptide comprises an amino acid sequencehaving at least 70% identity, at least 75% identity, at least 80%identity, preferably at least 85% identity, at least 90% identity, atleast 95% amino acid identity, at least 96% amino acid identity, atleast 97% amino acid identity, at least 98% amino acid identity, or atleast 99% amino acid sequence identity to any of SEQ ID NO: 1 or SEQ IDNO: 2 or SEQ ID NO: 3, and having at least a 80% of the biologicalactivity of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, respectively,with respect to inhibiting the Bax-NPM complex formation, or treating orpreventing AKI-mediated effects in a mouse model in vivo (see, themethods described in the Examples herein and FIG. 8).

In some embodiments, a NPM inhibitory peptide comprises amino acids78-103 of SEQ ID NO: 4, or amino acids 226-246 of SEQ ID NO: 4, or afragment or variant with at least 85% or 90% or 95% sequence identitythereto.

In some embodiments, a NPM inhibitory peptide comprises a fragment ofthe NPM polypeptide comprising any one or more of the phosphorylationsites of T86, S88, T95, T234 or S242. In some embodiments, a NPMinhibitory peptide comprises at least consecutive 10 amino acidsselected from any of amino acids 1-50, 40-60, 60-80, 60-120, 130-200,200-250, 220-250 of SEQ ID NO: 4. In some embodiments, if the NPMinhibitor peptide comprises any one of amino acids T86, S88, T95 of SEQID NO: 4, then each can exist as the phosphorylated forms (i.e., pT86,pS88, pT95). In alternative embodiments, if the NPM inhibitor peptidecomprises any one of amino acids T86, S88, T95 of SEQ ID NO: 4, theneach the can exist as the nonphosphorylated forms (i.e., nT86, nS88,nT95). In some embodiments, if the NPM inhibitor peptide comprises anyone of amino acids T234 or S242 of SEQ ID NO: 4, then the can each existas the non-phosphorylated forms (i.e., nT234 or nS242). In alternativeembodiments, if the NPM inhibitor peptide comprises any one of aminoacids T234 or S242 of SEQ ID NO: 4, then the can each exist as thephosphorylated forms (i.e., pT234 or pS242). It is envisioned that if anNPM inhibitory peptide comprises more than one phosphorylation aminoacid of T86, S88, T95, T234 or S242, the NPM inhibitory peptide cancomprise any combination of phosphorylation states.

In some embodiments of the compositions and methods described herein, aninhibitor of Bax-NPM complex formation comprises at least one NPMstructural analog, such as a dominant negative NPM peptide. Exemplarydominant negative NPM peptides are NPM (78-103) of SEQ ID NO: 4 orNPM(226-246) of SEQ ID NO: 4. In some embodiments, amino acids T86, S88and T95 of a NPM(78-103) peptide are phosphorylated. In someembodiments, amino acids T234 or S242 of a NPM(226-246) peptide arenon-phosphorylated. In some embodiments of the compositions and methodsdescribed herein, the RNA or DNA aptamer binds to or physicallyinteracts with or blocks the ischemic-induced phosphorylated form of theNPM polypeptide from binding to Bax.

In some embodiments of the compositions and methods described herein, aninhibitor of Bax-NPM complex formation comprises at least one solubleNPM peptide or fusion polypeptide thereof, such as, for example, a NPMinhibitory polypeptide. In some such embodiments, the NPM inhibitorypolypeptide is a dominant negative NPM fusion protein. In someembodiments of the compositions and methods described herein, the NPMinhibitory polypeptide comprises amino acids 78-103 of SEQ ID NO: 4 oramino acids 226-246 of SEQ ID NO: 4 of variants having at least 50%, or60%, or at least 70%, or at least 80%, or at least 90% or at least 95%or at least 98% sequence identity thereto.

B. Modifications of NPM Peptide Inhibitors

In some embodiments, a NPM inhibitory peptides of SEQ ID NO: 1-3 areconjugated or attached to renal targeting moieties or peptides,including renal targeting nuclear localization sequences (NLS), so thepeptides target the kidney. In some embodiments, a NPM peptide mimicfurther comprises a targeting sequence selected from the groupconsisting of: Ac-KKKRKV-(βA) (SEQ ID NO: 5), and AC-PKKKRKV-(βA) (SEQID NO: 6), or a variant at least 85% or at least 90% or at least 95%sequence identity to SEQ ID NO: 5 or 6. In some embodiments, the NPMinhibiting peptide can comprise a cell penetrating peptide (referred toherein as “CPP”) as disclosed in the Examples. Any CPP commonly known inthe art is envisioned for use, for example exemplary CPPs are disclosedin U.S. Pat. Nos. 7,579,318; 8,242,081; 8,372,951; 8,575,305; 8,044,019;2008/0234183, 2003/0229202; 2013/0164219, each of which are incorporatedherein in their entirety by reference.

A NPM inhibitory peptide disclosed herein, e.g. SEQ ID NO: 1-3 asdisclosed herein, may be synthesized by methods familiar to thoseskilled in the art or purchased commercially (#CG51068 RayBiotech, Inc.Norcross, Ga.).

Variations and modifications to a NPM inhibitory peptide can areenvisioned to provide means for targeting. For example, a NPM inhibitorypeptide as disclosed herein, can be linked with a molecularcounter-ligand, for example but not limited to, molecules which targetthe kidney, to make a NPM inhibitory peptide tissue specific.

In one embodiment, a NPM inhibitory peptide as disclosed herein islinked to a carrier to enhance its bioavailability. Such carriers areknown in the art and include poly (alkyl) glycol such as poly ethyleneglycol (PEG) or methoxypolyethylene glycol (mPEG) which can increase thein vivo half life of proteins to which they are conjugated. Methods ofPEGylation of a peptide are well known by one of ordinary skill in theart, and are considerations of, for example, how large a PEG polymer touse. In some embodiments, a peptide can be fused to serum albumin toincrease the serum half-life of therapeutic polypeptides and peptides.

It will be appreciated that a NPM inhibitory peptide as disclosedherein, useful in the methods and composition as disclosed herein canoptionally contain amino acids other than the 20 amino acids commonlyreferred to as the 20 naturally occurring amino acids.

In some embodiments, any of the amino acids of a NPM inhibitory peptideas disclosed herein, such as SEQ ID NO: 1-3 or a peptide having at least80% sequence identity thereto, including the terminal amino acids, canbe modified either by natural processes such as glycosylation and otherposttranslational modifications, or by chemical modification techniqueswhich are well known in the art. Even the common modifications thatoccur naturally in polypeptides are too numerous to list exhaustivelyhere, but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. Among the known modificationswhich can be present in polypeptides of the present invention are, toname an illustrative few, acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a polynucleotide or polynucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphotidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycation, glycosylation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, 1. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W.H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Sifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

It will also be appreciated, as is well known and as noted above, thatpeptides and polypeptides are not always entirely linear. For instance,polypeptides can be branched as a result of ubiquitination, and they canbe circular, with or without branching, generally as a result ofposttranslational events, including natural processing events and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides can be synthesizedby non translational natural processes and by entirely syntheticmethods.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring and;synthetic polypeptides and such modifications can be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylation host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylation asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification can be presentto the same or varying degree at several sites in a given polypeptide.Also, a given peptide or polypeptide can contain many types ofmodifications.

In some embodiments, N-methyl and hydroxy-amino acids can be substitutedfor conventional amino acids in solid phase peptide synthesis. However,production of polymers with reduced peptide bonds requires synthesis ofthe dimmer of amino acids containing the reduced peptide bond. Suchdimers are incorporated into polymers using standard solid phasesynthesis procedures. Other synthesis procedures are well known in theart.

Accordingly, functional derivatives of a NPM inhibitory peptide asdisclosed herein, may be prepared by modification of the amino acids ofSEQ ID NO: 1-3e are encompassed for use in the methods and compositionsas disclosed herein. Modifications may occur anywhere in the NPMinhibitory peptide as disclosed herein, e.g., a modification of anyamino acid in the sequences of any of SEQ ID NO; 1, SEQ ID NO: 2 or SEQID NO: 3 or its functional derivative polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.Modifications may include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of other functionalmoiety, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphotidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formylation, gamma-carboxylation, glycosylation,glycophosphatidylinositol (GPI) anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. See, for instance,E. Creighton Proteins-Structure and Molecular Properties, 2nd Ed., W. H.Freeman and Company, New York (1993); B. C. Johnson, Post TranslationalCovalent Modification of Proteins, Academic Press, New York, (1983);Seifter et al., Meth. Enzymol. 182: 626-646 (1990); Rattan et al., Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Preparation of these modifiedderivatives may, for example, be useful if direct administration of theGHK peptide is contemplated.

In some embodiments a NPM inhibitory peptide as disclosed herein, can beconjugated to a second entity, for example, to promote stability or forspecific cell type targeting. In some embodiments, a NPM inhibitorypeptide as disclosed herein, or fragments, derivatives or variantsthereof can be conjugated to a first fusion partner (i.e. IgG1 Fc). Theconjugation can be a non-covalent or covalent interaction, for example,by means of chemical crosslinkage or conjugation. As discussed herein,in some embodiments, a NPM inhibitory peptide as disclosed herein, isfused to serum albumin to increase the serum half-life of the a NPMinhibitory peptide, e.g., SEQ ID NO: 1-3.

In some embodiments, a NPM inhibitory peptide as disclosed herein, canalso be fused to a second fusion partner, for example, to a polypeptidethat targets the product to a desired location, or, for example, a tagthat facilitates its purification, if so desired. Tags and fusionpartners can be designed to be cleavable, if so desired. Anothermodification specifically contemplated is attachment, e.g., covalentattachment, to a polymer. In one aspect, polymers such as polyethyleneglycol (PEG) or methoxypolyethylene glycol (mPEG) can increase the invivo half-life of proteins to which they are conjugated. Methods ofPEGylation of polypeptide agents are well known to those skilled in theart, as are considerations of, for example, how large a PEG polymer touse.

As used herein, the term “conjugate” or “conjugation” refers to theattachment of two or more entities to form one entity. For example, themethods of the present invention provide conjugation of a NPM inhibitorypeptide as disclosed herein, or fragments, derivatives or variantsthereof joined with another entity, for example a moiety such as a firstfusion partner that makes the NPM inhibitory peptide stable, such as Igcarrier particle, for example IgG1 Fc. The attachment can be by means oflinkers, chemical modification, peptide linkers, chemical linkers,covalent or non-covalent bonds, or protein fusion or by any means knownto one skilled in the art. The joining can be permanent or reversible.In some embodiments, several linkers can be included in order to takeadvantage of desired properties of each linker and each protein in theconjugate. Flexible linkers and linkers that increase the solubility ofthe conjugates are contemplated for use alone or with other linkers asdisclosed herein. Peptide linkers can be linked by expressing DNAencoding the linker to one or more proteins in the conjugate. Linkerscan be acid cleavable, photocleavable and heat sensitive linkers.Methods for conjugation are well known by persons skilled in the art andare encompassed for use in the present invention.

According to the present invention, a NPM inhibitory peptide asdisclosed herein, or fragments, derivatives or variants thereof, can belinked to the first fusion partner via any suitable means, as known inthe art, see for example U.S. Pat. Nos. 4,625,014, 5,057,301 and5,514,363, which are incorporated herein in their entirety by reference.For example, the GHK peptide e can be covalently conjugated to the IgG1Fc, either directly or through one or more linkers. In one embodiment, aNPM inhibitory peptide as disclosed herein is conjugated directly to thefirst fusion partner (e.g. Fc), and in an alternative embodiment, a NPMinhibitory peptide as disclosed herein can be conjugated to a firstfusion partner (such as IgG1 Fc) via a linker, e.g. a transportenhancing linker.

A large variety of methods for conjugation of a NPM inhibitory peptideas disclosed herein, with a first fusion partner (e.g. Fc) are known inthe art. Such methods are e.g. described by Hermanson (1996,Bioconjugate Techniques, Academic Press), in U.S. Pat. Nos. 6,180,084and 6,264,914 which are incorporated herein in their entirety byreference and include e.g. methods used to link haptens to carriersproteins as routinely used in applied immunology (see Harlow and Lane,1988, “Antibodies: A laboratory manual”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). It is recognized that, in some cases,a NPM inhibitory peptide as disclosed herein can lose efficacy orfunctionality upon conjugation depending, e.g., on the conjugationprocedure or the chemical group utilized therein. However, given thelarge variety of methods for conjugation the skilled person is able tofind a conjugation method that does not or least affects the efficacy orfunctionality of the entities, such as the NPM inhibitory peptide to beconjugated.

Suitable methods for conjugation of a NPM inhibitory peptide asdisclosed herein with a first fusion partner (e.g. Fc) include e.g.carbodimide conjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70:151-159). Alternatively, a moiety can be coupled to a targeting agent asdescribed by Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273(1996), and Nagy et al., Proc. Natl. Acad. Sci. USA 95:1794-1799 (1998),each of which are incorporated herein by reference. Another method forconjugating one can use is, for example sodium periodate oxidationfollowed by reductive alkylation of appropriate reactants andglutaraldehyde crosslinking.

One can use a variety of different linkers to conjugate a NPM inhibitorypeptide as disclosed herein, with a first fusion partner (e.g. Fc), forexample but not limited to aminocaproic horse radish peroxidase (HRP) ora heterobiofunctional cross-linker, e.g. carbonyl reactive andsulfhydryl-reactive cross-linker Heterobiofunctional cross linkingreagents usually contain two reactive groups that can be coupled to twodifferent function targets on proteins and other macromolecules in a twoor three-step process, which can limit the degree of polymerizationoften associated with using homobiofunctional cross-linkers. Suchmulti-step protocols can offer a great control of conjugate size and themolar ratio of components.

The term “linker” refers to any means to join two or more entities, forexample a NPM inhibitory peptide as disclosed herein, with a firstfusion partner (e.g. Fc). A linker can be a covalent linker or anon-covalent linker Examples of covalent linkers include covalent bondsor a linker moiety covalently attached to one or more of the proteins tobe linked. The linker can also be a non-covalent bond, e.g. anorganometallic bond through a metal center such as platinum atom. Forcovalent linkages, various functionalities can be used, such as amidegroups, including carbonic acid derivatives, ethers, esters, includingorganic and inorganic esters, amino, urethane, urea and the like. Toprovide for linking, the effector molecule and/or the probe can bemodified by oxidation, hydroxylation, substitution, reduction etc. toprovide a site for coupling. It will be appreciated that modificationwhich do not significantly decrease the function of the a NPM inhibitorypeptide as disclosed herein or the first fusion partner (e.g. Fc) arepreferred.

The dosage ranges for the administration of a NPM inhibitory peptide asdisclosed herein, e.g., a peptide of SEQ ID NO: 1-3 or a peptide with atleast 80% sequence identity thereto depend upon the form of the protein,and its potency, as described further herein, and are amounts largeenough to produce the desired effect in which the symptoms, markers, orsigns of kidney disease or AKI are reduced. The dosage should not be solarge as to cause adverse side effects. Generally, the dosage can varywith the age, condition, and sex of the patient and can be determined byone of skill in the art. The dosage can also be adjusted by theindividual physician in the event of any complication. Typically, thedosage ranges from 0.001 mg/kg body weight to 1000 mg/kg body weight. Inone embodiment, the dose range is from 0.5 μg/kg body weight to 25 mg/kgbody weight. The doses can be given once a day, less than once a day ormultiple times a day in order to achieve a therapeutically effectivedose.

With respect to the therapeutic methods of the invention, it is notintended that the administration of a NPM inhibitory peptide asdisclosed herein, be limited to a particular mode of administration,dosage, or frequency of dosing; the present invention contemplates allmodes of administration, including intramuscular, intravenous,inhalation, intraperitoneal, intravesicular, intraarticular,intralesional, subcutaneous, or any other route sufficient to provide adose adequate to treat the ischemic injury and/or kidney injury or AKI.The therapeutic may be administered to the patient in a single dose orin multiple doses. When multiple doses are administered, the doses maybe separated from one another by, for example, one hour, three hours,six hours, eight hours, one day, two days, one week, two weeks, or onemonth. For example, the therapeutic may be administered for, e.g., 2, 3,4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that,for any particular subject, specific dosage regimes should be adjustedovertime according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions. For example, the dosage of the therapeutic can beincreased if the lower dose does not provide sufficient therapeuticactivity.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, therapeutically effective amounts of a NPMinhibitory peptide as disclosed herein, can be provided at a dose of0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test bioassays or systems.

Dosages for a particular patient or subject can be determined by one ofordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Aphysician may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. The dose administered to a patient is sufficient to effect abeneficial therapeutic response in the patient over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of the NPMinhibitory peptide as disclosed herein, or functional derivativesthereof, and the condition of the patient, as well as the body weight orsurface area of the patient to be treated. The size of the dose is alsodetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,formulation, or the like in a particular subject. Therapeuticcompositions comprising a NPM inhibitory peptide as disclosed herein, orfunctional derivatives thereof are optionally tested in one or moreappropriate in vitro and/or in vivo animal models of disease, such asmice exposed to cigarette smoke (Shapiro Chest 2000 117:2235-75), toconfirm efficacy, tissue metabolism, and to estimate dosages, accordingto methods well known in the art. In particular, dosages can beinitially determined by activity, stability or other suitable measuresof treatment vs. non-treatment (e.g., comparison of treated vs.untreated cells or animal models), in a relevant assay. Formulations areadministered at a rate determined by the LD50 of the relevantformulation, and/or observation of any side-effects of the NPMinhibitory peptide as disclosed herein, or functional derivativesthereof at various concentrations, e.g., as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses.

In determining the effective amount of a NPM inhibitory peptide asdisclosed herein, or functional derivatives thereof to be administeredin the treatment or prophylaxis of disease the physician evaluatescirculating plasma levels, formulation toxicities, and progression ofthe disease.

C. Pharmaceutical Compositions and Modes of Administration

An inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein can be administered to a subject in need thereof by anyappropriate route which results in an effective treatment in thesubject. As used herein, the terms “administering,” and “introducing”are used interchangeably and refer to the placement of an inhibitor ofBax-NPM complex formation or a NPM peptide inhibitor, as describedherein into a subject by a method or route which results in at leastpartial localization of such agents at a desired site, such that adesired effect(s) is produced.

In some embodiments, an inhibitor of Bax-NPM complex formation or a NPMpeptide inhibitor, as described herein is administered to a subjecthaving a chronic kidney disease by any mode of administration thatdelivers the agent systemically or to a desired surface or target, andcan include, but is not limited to, injection, infusion, instillation,and inhalation administration. To the extent that polypeptide agents canbe protected from inactivation in the gut, oral administration forms arealso contemplated. “Injection” includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, aninhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein for use in the methods described herein areadministered by intravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally”as used herein, refer to modes of administration other than enteral andtopical administration, usually by injection. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer tothe administration of an inhibitor of Bax-NPM complex formation or a NPMpeptide inhibitor, as described herein, other than directly into atarget site, tissue, or organ, such as a tumor site, such that it entersthe subject's circulatory system and, thus, is subject to metabolism andother like processes.

For the clinical use of the methods described herein, administration ofan inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein, can include formulation into pharmaceuticalcompositions or pharmaceutical formulations for parenteraladministration, e.g., intravenous; mucosal, e.g., intranasal; ocular, orother mode of administration. In some embodiments, an inhibitor ofBax-NPM complex formation or a NPM peptide inhibitor, as describedherein can be administered along with any pharmaceutically acceptablecarrier compound, material, or composition which results in an effectivetreatment in the subject. Thus, a pharmaceutical formulation for use inthe methods described herein can contain an inhibitor of Bax-NPM complexformation or a NPM peptide inhibitor, as described herein, as describedherein, in combination with one or more pharmaceutically acceptableingredients.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically acceptablecarrier” as used herein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, media, encapsulating material, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in maintaining thestability, solubility, or activity of, an inhibitor of Bax-NPM complexformation or a NPM peptide inhibitor, as described herein. Each carriermust be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such ascocoa butter and suppository waxes; (8) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (9) glycols, such as propylene glycol; (10) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11)esters, such as ethyl oleate and ethyl laurate; (12) agar; (13)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17)Ringer's solution; (19) pH buffered solutions; (20) polyesters,polycarbonates and/or polyanhydrides; (21) bulking agents, such aspolypeptides and amino acids (22) serum components, such as serumalbumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24)other non-toxic compatible substances employed in pharmaceuticalformulations. Release agents, coating agents, preservatives, andantioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

An inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein can be specially formulated for administration of thecompound to a subject in solid, liquid or gel form, including thoseadapted for the following: (1) parenteral administration, for example,by subcutaneous, intramuscular, intravenous or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation; (2) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (3)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (4) ocularly; (5) transdermally; (6) transmucosally; or (79)nasally. Additionally, an inhibitor of Bax-NPM complex formation or aNPM peptide inhibitor, as described herein can be implanted into apatient or injected using a drug delivery system. See, for example,Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984);Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals”(Plenum Press, New York, 1981); U.S. Pat. Nos. 3,773,919; and 353,270,960.

In some embodiments, an inhibitor of Bax-NPM complex formation or a NPMpeptide inhibitor as disclosed herein is administered within 48 hrs ofan ischemic insult or injury, or within 36 hours, or 24 hours, or 12hrs, or within 6 hours of an ischemic insult or injury. In someembodiments, an inhibitor of Bax-NPM complex formation or a NPM peptideinhibitor as disclosed herein is administered within 0-3 hours, or 0-6hours of an ischemic insult or injury, or within 6-12 hrs, or 12-24 hrsof an ischemic injury or insult.

Further embodiments of the formulations and modes of administration ofthe compositions comprising an inhibitor of Bax-NPM complex formation ora NPM peptide inhibitor, as described herein, that can be used in themethods described herein are described below.

Parenteral Dosage Forms.

Parenteral dosage forms of an inhibitor of Bax-NPM complex formation ora NPM peptide inhibitor, as described herein can also be administered toa subject with a chronic kidney condition by various routes, including,but not limited to, subcutaneous, intravenous (including bolusinjection), intramuscular, and intraarterial. Since administration ofparenteral dosage forms typically bypasses the patient's naturaldefenses against contaminants, parenteral dosage forms are preferablysterile or capable of being sterilized prior to administration to apatient. Examples of parenteral dosage forms include, but are notlimited to, solutions ready for injection, dry products ready to bedissolved or suspended in a pharmaceutically acceptable vehicle forinjection, suspensions ready for injection, controlled-releaseparenteral dosage forms, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe disclosure are well known to those skilled in the art. Examplesinclude, without limitation: sterile water; water for injection USP;saline solution; glucose solution; aqueous vehicles such as but notlimited to, sodium chloride injection, Ringer's injection, dextroseInjection, dextrose and sodium chloride injection, and lactated Ringer'sinjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and propylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

In some embodiments, compositions comprising an effective amount of aninhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein are formulated to be suitable for oral administration,for example as discrete dosage forms, such as, but not limited to,tablets (including without limitation scored or coated tablets), pills,caplets, capsules, chewable tablets, powder packets, cachets, troches,wafers, aerosol sprays, or liquids, such as but not limited to, syrups,elixirs, solutions or suspensions in an aqueous liquid, a non-aqueousliquid, an oil-in-water emulsion, or a water-in-oil emulsion. Suchcompositions contain a predetermined amount of the pharmaceuticallyacceptable salt of the disclosed compounds, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton, Pa. (1990).

Due to their ease of administration, tablets and capsules represent themost advantageous solid oral dosage unit forms, in which case solidpharmaceutical excipients are used. If desired, tablets can be coated bystandard aqueous or nonaqueous techniques. These dosage forms can beprepared by any of the methods of pharmacy. In general, pharmaceuticalcompositions and dosage forms are prepared by uniformly and intimatelyadmixing the active ingredient(s) with liquid carriers, finely dividedsolid carriers, or both, and then shaping the product into the desiredpresentation if necessary. In some embodiments, oral dosage forms arenot used for the antibiotic agent.

Typical oral dosage forms of the compositions comprising an effectiveamount of an inhibitor of Bax-NPM complex formation or a NPM peptideinhibitor, as described herein are prepared by combining thepharmaceutically acceptable salt of the inhibitor of Bax-NPM complexformation or the NPM peptide inhibitor, as described herein in anintimate admixture with at least one excipient according to conventionalpharmaceutical compounding techniques. Excipients can take a widevariety of forms depending on the form of the composition desired foradministration. For example, excipients suitable for use in oral liquidor aerosol dosage forms include, but are not limited to, water, glycols,oils, alcohols, flavoring agents, preservatives, and coloring agents.Examples of excipients suitable for use in solid oral dosage forms(e.g., powders, tablets, capsules, and caplets) include, but are notlimited to, starches, sugars, microcrystalline cellulose, kaolin,diluents, granulating agents, lubricants, binders, and disintegratingagents.

Binders suitable for use in the pharmaceutical formulations describedherein include, but are not limited to, corn starch, potato starch, orother starches, gelatin, natural and synthetic gums such as acacia,sodium alginate, alginic acid, other alginates, powdered tragacanth,guar gum, cellulose and its derivatives (e.g., ethyl cellulose,cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinizedstarch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical formulationsdescribed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof. The binder orfiller in pharmaceutical compositions described herein is typicallypresent in from about 50 to about 99 weight percent of thepharmaceutical composition.

Disintegrants are used in the oral pharmaceutical formulations describedherein to provide tablets that disintegrate when exposed to an aqueousenvironment. A sufficient amount of disintegrant that is neither toolittle nor too much to detrimentally alter the release of the activeingredient(s) should be used to form solid oral dosage forms of aninhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein. The amount of disintegrant used varies based upon thetype of formulation, and is readily discernible to those of ordinaryskill in the art. Disintegrants that can be used to form oralpharmaceutical formulations include, but are not limited to, agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used to form oral pharmaceutical formulations ofan inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein, include, but are not limited to, calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, stearic acid, sodiumlauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL® 200, manufactured by W. R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Piano, Tex.), CAB-O-SIL® (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

In other embodiments, lactose-free pharmaceutical formulations anddosage forms are provided, wherein such compositions preferably containlittle, if any, lactose or other mono- or di-saccharides. As usedherein, the term “lactose-free” means that the amount of lactosepresent, if any, is insufficient to substantially increase thedegradation rate of an active ingredient. Lactose-free compositions ofthe disclosure can comprise excipients which are well known in the artand are listed in the USP (XXI)/NF (XVI), which is incorporated hereinby reference.

The oral formulations of an inhibitor of Bax-NPM complex formation or aNPM peptide inhibitor, as described herein further encompass, in someembodiments, anhydrous pharmaceutical compositions and dosage formscomprising the ROBO2 inhibitors described herein as active ingredients,since water can facilitate the degradation of some compounds. Forexample, the addition of water (e.g., 5%) is widely accepted in thepharmaceutical arts as a means of simulating long-term storage in orderto determine characteristics such as shelf life or the stability offormulations overtime. See, e.g., Jens T. Carstensen, Drug Stability:Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995).Anhydrous pharmaceutical compositions and dosage forms described hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. Anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials)with or without desiccants, blister packs, and strip packs.

Aerosol Formulations.

An inhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein can be packaged in a pressurized aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. Aninhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein can also be administered in a non-pressurized form suchas in a nebulizer or atomizer. An inhibitor of Bax-NPM complex formationor a NPM peptide inhibitor, as described herein inhibitor can also beadministered directly to the airways in the form of a dry powder, forexample, by use of an inhaler.

Suitable powder compositions include, by way of illustration, powderedpreparations of an inhibitor of Bax-NPM complex formation or a NPMpeptide inhibitor, as described herein, thoroughly intermixed withlactose, or other inert powders acceptable for intrabronchialadministration. The powder compositions can be administered via anaerosol dispenser or encased in a breakable capsule which can beinserted by the subject into a device that punctures the capsule andblows the powder out in a steady stream suitable for inhalation. Thecompositions can include propellants, surfactants, and co-solvents andcan be filled into conventional aerosol containers that are closed by asuitable metering valve.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev.Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S, and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

The formulations of an inhibitor of Bax-NPM complex formation or a NPMpeptide inhibitor, as described herein further encompass anhydrouspharmaceutical compositions and dosage forms comprising the disclosedcompounds as active ingredients, since water can facilitate thedegradation of some compounds. For example, the addition of water (e.g.,5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf life or the stability of formulations overtime. See, e.g., JensT. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed.,Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositionsand dosage forms of the disclosure can be prepared using anhydrous orlow moisture containing ingredients and low moisture or low humidityconditions. Pharmaceutical compositions and dosage forms that compriselactose and at least one active ingredient that comprises a primary orsecondary amine are preferably anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected. Anhydrous compositions are preferably packaged usingmaterials known to prevent exposure to water such that they can beincluded in suitable formulary kits. Examples of suitable packaginginclude, but are not limited to, hermetically sealed foils, plastics,unit dose containers (e.g., vials) with or without desiccants, blisterpacks, and strip packs.

Controlled and Delayed Release Dosage Forms.

In some embodiments of the aspects described herein, an inhibitor ofBax-NPM complex formation or a NPM peptide inhibitor, as describedherein can be administered to a subject by controlled- ordelayed-release means. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. (Kim, Chemg-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)).Controlled-release formulations can be used to control a compound offormula (I)'s onset of action, duration of action, plasma levels withinthe therapeutic window, and peak blood levels. In particular,controlled- or extended-release dosage forms or formulations can be usedto ensure that the maximum effectiveness of a compound of formula (I) isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the ROBO-2inhibitors described herein. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each ofwhich is incorporated herein by reference in their entireties. Thesedosage forms can be used to provide slow or controlled-release of one ormore active ingredients using, for example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)),multilayer coatings, microparticles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Additionally, ion exchange materials can be used to prepareimmobilized, adsorbed salt forms of the disclosed compounds and thuseffect controlled delivery of the drug. Examples of specific anionexchangers include, but are not limited to, DUOLITE® A568 and DUOLITE®AP143 (Rohm & Haas, Spring House, Pa. USA).

In some embodiments of the methods described herein, an inhibitor ofBax-NPM complex formation or a NPM peptide inhibitor, as describedherein for use in the methods described herein is administered to asubject by sustained release or in pulses. Pulse therapy is not a formof discontinuous administration of the same amount of a composition overtime, but comprises administration of the same dose of the compositionat a reduced frequency or administration of reduced doses. Sustainedrelease or pulse administrations are particularly preferred when thedisorder occurs continuously in the subject, for example where thesubject has chronic kidney disease. Each pulse dose can be reduced andthe total amount of a ROBO-2 inhibitor described herein administeredover the course of treatment to the subject or patient is minimized.

The interval between pulses, when necessary, can be determined by one ofordinary skill in the art. Often, the interval between pulses can becalculated by administering another dose of the composition when thecomposition or the active component of the composition is no longerdetectable in the subject prior to delivery of the next pulse. Intervalscan also be calculated from the in vivo half-life of the composition.Intervals can be calculated as greater than the in vivo half-life, or 2,3, 4, 5 and even 10 times greater the composition half-life. Variousmethods and apparatus for pulsing compositions by infusion or otherforms of delivery to the patient are disclosed in U.S. Pat. Nos.4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.

In some embodiments, sustained-release preparations comprising aninhibitor of Bax-NPM complex formation or a NPM peptide inhibitor, asdescribed herein can be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the inhibitor, in which matrices are in the form ofshaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations comprising an inhibitor of Bax-NPM complex formation ora NPM peptide inhibitor, as described herein to be used for in vivoadministration are preferably sterile. This is readily accomplished byfiltration through, for example, sterile filtration membranes, and othermethods known to one of skill in the art.

IV. Diagnostic Assays for Detection of AKI

In one aspect, the invention provides for methods, compositions and kitsfor detecting ischemic-stress induced phosphorylation status of NPM. Inparticular, one aspect relates to a diagnostic assay, method andcomposition to assess if a subject has AKI, wherein the method comprisesassessing the phosphorylation status of the NPM polypeptide, anddetecting a change of at least one of the phosphorylation sites asfollows: T86, S88, T95, T234 or S242. In particular, under normalconditions (e.g., non-stress conditions) T86, S88, T95 of the NPMpolypeptide are unphosphorylated, and become phosphorylated understressful conditions to become phospho-T86, phospho-S88, phospho-T95. Incontrast, under normal conditions (e.g., non-stress conditions) T234 andS242 of the NPM polypeptide are phosphorylated, and becomedephosphorylated under stressful conditions. That is, under normalconditions, the phosphorylation state of the NPM polypeptide is T86,S88, T95, phospho-T234, phospho-S242, and under stressful conditions, orafter AKI, the phosphorylation state of the NPM polypeptide isphospho-T86, phospho-S88, phospho-T95, T234, S242.

Accordingly, in some embodiments, an assay can comprise a method todetect at least one or more of: phospho-T86, phospho-S88, phospho-T95,unphosporylated-T234 and unphosporylated-S242 of a NPM polypeptide in abiological sample obtained from a subject, wherein detection of at leastone, or at least 2, or at least 3 or at least 4 of the abovephosphorylation states of NPM polypeptide identifies a subject withkidney injury or AKI. Detection of the phosphorylation states can be byany means, e.g., mass spectrometry, antibodies or antibody fragments,including but not limited to, pan-specific phospho-Ser (anti-pSer) orphospho-Thr (anti-pThr) antibodies, as well as phospho-specificantibodies, e.g., anti-phospho-T86, anti-phospho-S88, anti-phospho-T95,anti-phospho-T234 and anti-phospho-S242 antibodies, or antibodyfragments or antigen binding fragments thereof.

Exemplary biological samples include, but are not limited to, a kidneybiopsy sample, serum, blood, plasma and urine. Additionally, theinventors have also discovered that interruption of the stress-inducedNPM phosphorylation events, e.g., using one of three different blockingpeptides, can decrease cell death in the kidney due to metabolic stress(including ischemic stress and hypoxic stress), and can be used astreatment, including therapeutic treatment to treat a subject with AKI,or alternatively, as a prophylactic treatment to prevent the subjectdeveloping AKI.

A. Stress- and Ischemia-Induced Phosphorylation States of NPM

In one aspect, the invention provides a method for diagnosing kidneyinjury, e.g., acute kidney injury (AKI) in a subject by measuring theischemic-induced, or stress-induced phosphorylation of NPM polypeptidein a biological sample, e.g., a blood sample or urine sample obtainedfrom the subject. As discussed herein, under normal (non-stressful)conditions, the phosphorylation state of the NPM polypeptide is T86,S88, T95, phospho-T234, phospho-S242. In contrast, under stressfulconditions, or after AKI, the phosphorylation state of the NPMpolypeptide changes to phospho-T86, phospho-S88, phospho-T95, T234,S242. That is, after ischemic or hypoxic stress, one or more of aminoacid residues T86, S88, T95 of the NPM polypeptide becomephosphorylated, and one or both of phospho-T234, phospho-S242 on the NPMpolypeptide become dephosphorylated (to T234 and S242). It is envisionedthat not all these phosphorylation events necessarily occur underischemic or stress-induced conditions as described herein in theExamples, and therefore, in some embodiments, at least 2, or at least 3,or at least 4 or at least 5 of the phosphorylation events can bemeasured.

For simplicity purposes herein, non-phosphorylated Ser or Thr residuesare represented as nS or nT respectively. Therefore, the stress-inducedphosphorylation status of NPM can be represented as having at least 1 orat least 2, or at least 3, or at least 4 or at least 5 of the followingphosphorylation states: pT86, pS88, pT95, nT234, nS242, and the undernormal conditions, the phosphorylation status of NPM can be representedas having at least 1 or at least 2, or at least 3, or at least 4 or atleast 5 of the following phosphorylation states nT86, nS88, nT95, pT234,pS242.

In some embodiments, one can measure at least one, or at least 2 or atleast 3, or at least 4 or at least all 5 phosphorylation changes, e.g.,at least one, or at least 2 or at least 3, or at least 4 or at least all5 phosphorylation changes of T86 to pT86, S88 to pS88, T95 to pT95,pT234 to T234, pS242 to S242.

In some embodiments, one can measure at least one, or at least 2 or allthree of: T86 to pT86, S88 to pS88, T95 to pT95, and also measure atleast one of pT234 to T234 and/or pS242 to S242.

In some embodiments, if in a biological sample obtained from a subject,at least one of, or at least two of, or at least three of: pT86, pS88,pT95 is detected, and at least one of: nT234 or nS242 is detected, thenthe subject can be selected for having, or at risk of having kidneyinjury, including but not limited to AKI, or can be selected as havingischemia or organ toxicity.

In some embodiments, if in a biological sample obtained from a subject,at least one of, or at least two of, or at least three of, at least 4 ofor at least 5 of: pT86, pS88, pT95, nT234, nS242 is detected, then thesubject can be selected for having, or at risk of having kidney injury,including but not limited to AKI, or can be selected as having ischemiaor organ toxicity. Any combination is envisioned, for example, Table 1shows exemplary combinations of different phosphorylation states of NPMin a biological sample from a subject can be used to select as having,or at risk of having kidney injury, including but not limited to AKI, orcan be selected as having ischemia or organ toxicity.

TABLE 1 Exemplary combinations of phosphorylation states of the NPMpolypeptide that can be detected in a method or assay that identify asubject, (or can be used to select a subject) as having, or risk ofhaving kidney injury, AKI, ischemia or organ toxicity herein. “X” showsany combination of measurement of at least one, or at least 2 or atleast 3, or at least 4 or at least all 5 phosphorylation states of pT86,pS88, pT95, nT234, and nS242 (where nT234 and nS242 identifies non-phosphorylated T234 and S242 respectively). pT86 pS88 pT95 nT234 nS242 XX X X X X X X X X X X X X X X X X X X x X X X X X X X X X X X X X X X XX X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X XX X X X x X X

In another embodiment, the methods disclosed herein can be used tomeasure a combination of at least one, or at least 2, or at least 3, orat least 4 or at least 5 of phosphorylation states of NPM polypeptide asshown in Table 2, where if their presence is detected, it identifies asubject, or can be used to select a subject, as having, or risk ofhaving kidney injury, AKI, ischemia or organ toxicity as describedherein

TABLE 2 Exemplary combinations of phosphorylation states of the NPMpolypeptide assessed in a method or assay to identify a subject, (or canbe used to select a subject) as having, or risk of having kidney injury,AKI, ischemia or organ toxicity as described herein. Combi- Combo ofCombo of Combo of All five nation any 2/5 any 3/5 any 4/5 NPM of NPMphos- NPM phos- NPM phos- phos- any one phorylation phorylationphorylation phorylation maker sites sites sites sites pT86 pT86, pS88pT86, pS88, pT86, pS88, pT86, pS88, pT95 pT95, nT234 pT95, nT234, nS242pS88 pT86, pT95 pT86, pS88, pT86, pS88, nT234 pT95, nS242 pT95 pT86,nT234 pT86, pS88, pT86, pS88, nS242 nT234, nS242 nT234 pT86, nS242 pT86,pT95, pT86, pT95, nT234 nT234, nS242 nS242 pS88, pT95 pT86, pT95, pS88,pT95, nS242 nT234, nS242 pS88, nT234 pT86, nT234, nS242 pS88, nS242pS88, pT95, nT234 pT95, nT234 pS88, pT95, nS242 pT95, nS242 pS88, nT234,nS242 nT234, nS242 pT95, nT234, nS242

In alternative embodiments, if in a biological sample obtained from asubject, at least three of, at least 4 of or at least 5 of: nT86, nS88,nT95, pT234, pS242 is detected, then the subject is not selected oridentified as having, or at risk of having kidney injury, including butnot limited to AKI, or is not selected as having ischemia or organtoxicity. For example, in such an embodiment, the methods disclosedherein can be used to measure a combination of at least 3, or at least 4or at least 5 of phosphorylation states of NPM polypeptide as shown inTable 3, where if their presence is detected, it identifies a subject,or can be used to select a subject, as not having, or not at risk ofhaving kidney injury, AKI, ischemia or organ toxicity.

TABLE 3 Exemplary combinations of phosphorylation states of the NPMpolypeptide assessed in a method or assay to identify a subject, or toselect a subject, as not having, or not risk of having kidney injury,AKI, ischemia or organ toxicity. Combo of any 3/5 Combo of any 4/5 Allfive NPM NPM phos- NPM phos- phosphorylation phorylation phorylationsites sites sites nT86, nS88, nT95 nT86, nS88, nT95, nT86, nS88, nT95,pT234 pT234, pS242 nT86, nS88, pT234 nT86, nS88, nT95, pS242 nT86, nS88,pS242 nT86, nS88, pT234, pS242 nT86, nT95, pT234 nT86, nT95, pT234,pS242 nT86, nT95, pS242 nS88, nT95, pT234, pS242 nT86, pT234, pS242nS88, nT95, pT234 nS88, nT95, pS242 nS88, pT234, pS242 nT95, pT234,pS242

In some embodiments, the diagnostic method comprises measuring anddetecting the phosphorylation of at least one of serine or threonineresidue selected from: T86, S88, or T95 of the nucleophosmin (NPM)polypeptide; or the absence of phosphorylation of at least one of atleast one serine residue selected from T234 or S242 on a nucleophosmin(NPM) polypeptide.

In some embodiments, the diagnostic method comprises measuring anddetecting, in a biological sample obtained from the subject, at leastone of: the presence of phosphorylation of at least one of serine orthreonine residue selected from: T86, S88, or T95 of the nucleophosmin(NPM) polypeptide; or the absence of phosphorylation of at least one ofat least one serine residue selected from T234 or S242 on anucleophosmin (NPM) polypeptide.

In some embodiments, a method to determine if a subject has kidneyinjury or acute kidney injury (AKI), comprises (a) using an assay todetect at least on of: (i) the presence of phosphorylation of at leastone of serine or threonine residue selected from: T86, S88, or T95 ofthe nucleophosmin (NPM) polypeptide in a biological sample obtained froma subject; and/or (ii) the absence of phosphorylation of at least one ofat least one serine residue selected from T234 or S242 on anucleophosmin (NPM) polypeptide; and (b) selecting the subject as havingkidney injury or acute kidney injury (AKI), if the subject has thepresence of phosphorylation of at least one of: T86, S88, or T95 of thenucleophosmin (NPM) polypeptide is detected, or the absence ofphosphorylation of at least one of at least one serine residue selectedfrom T234 or S242 on a nucleophosmin (NPM) polypeptide, or both. Forexample, in some embodiments, one can use antibodies or antigen-bindingfragments thereof which specifically bind to any one or more of pT86,pS88, or pT95, and/or pT234 or pS242, and where specific binding of oneor more antibody or antigen-binding fragment to pT86, pS88, or pT95 isdetected, and binding is not detected with the antibodies orantigen-binding fragments to pT234 or pS242, it identifies a subject tohave kidney injury, and in some embodiments, AKI, or other ischemictissue stress, or organ injury.

In some embodiments, the method further comprises administering aneffective amount of a treatment for kidney injury or AKI to the subjectdiagnosed and selected as having kidney injury or acute kidney injury(AKI) in step (b). In some embodiments, an effective amount of atreatment for kidney injury or AKI is administering to the subject anagent which inhibits the phosphorylation of at least one of: T86, S88,T95 of the NPM polypeptide, and/or inhibits the dephsporylation of atleast one of: T232 or S240 of the NPM polypeptide, thereby inhibitingthe formation of a NPM-Bax complex. In alternative embodiments, aneffective amount of a treatment for kidney injury or AKI isadministering to the subject wherein the treatment for kidney injury orAKI is administering to a subject a composition comprising at least onepeptide from any peptide comprising the amino acid sequences of:TVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1); TLKMSVQPTVSLGGFEITPPVVLRLK (SEQ IDNO: 2) and ESFKKQEKTPKTPKGPSSVEDIKAK (SEQ ID NO: 3), or peptide with atleast 85% or 90% or 95% sequence identity to any of SEQ ID NO: 1-3.

In some embodiments, a method to determine if a subject has kidneyinjury or acute kidney injury (AKI), comprises obtaining a biologicalsample from a subject, and measuring for the presence of phosphorylationof at least one serine or threonine residue on the nucleophosmin (NPM)polypeptide at residues T86, S88, or T95, or measuring the absence ofphosphorylation of at least one serine residue T234 and S242 on thenucleophosmin (NPM) polypeptide, or both.

In some embodiments, a method comprises obtaining a biological samplefrom a subject, and measuring for the presence of phosphorylation of atleast one serine or threonine residue on the nucleophosmin (NPM)polypeptide at residues T86, S88, or T95, and measuring the absence ofphosphorylation of at least one serine residue T234 and S242 on thenucleophosmin (NPM) polypeptide.

The diagnostic methods, assays and kits can be performed on anybiological sample obtained from the subject, including but not limitedto urine samples, blood samples, biopsy samples and the like. In someembodiments, the sample is selected from the group consisting of; awhole blood sample a plasma sample, a serum sample or a fractionatedblood sample.

In some embodiments, the presence of phosphorylation of at least oneresidues on the NPM polypeptide at residues T86, S88, or T95 isdetermined by measuring and detecting the presence of binding of aphosphorylation specific antibody that preferentially bind to any oneof: phospho-T86, phopho-S88 or phospho-T95 on the NPM polypeptide. Insome embodiments, the absence of phosphorylation of at least one serineresidue T234 and S242 on NPM polypeptide is determined by measuring anddetecting the absence of binding of a phosphorylation specific antibodythat preferentially bind to any one of: phospho-T234 or phopho-S242 onthe NPM polypeptide.

In some embodiments, the presence of phosphorylation of at least oneresidues on the NPM polypeptide at residues T86, S88, or T95 isdetermined by measuring the presence of binding with at least one of:(i) a phosphorylation specific antibody or antigen-binding fragmentthereof that specifically binds to phospho-T86 on the NPM polypeptide;(ii) a phosphorylation specific antibody or antigen-binding fragmentthereof that specifically binds to phospho-S886 on the NPM polypeptide;or (iii) a phosphorylation specific antibody or antigen-binding fragmentthereof that specifically binds to phospho-T95 on the NPM polypeptide.

In some embodiments, the absence of phosphorylation of at least oneserine residue or threonine residue selected from T234 and S242 on theNPM polypeptide is determined by the absence of binding with at leastone of: (i) a phosphorylation specific antibody or antigen-bindingfragment thereof that specifically binds to phospho-T234 on the NPMpolypeptide; or (ii) a phosphorylation specific antibody orantigen-binding fragment thereof that specifically binds to phospho-S242on the NPM polypeptide.

B. Diagnosis of Kidney Injury

In some embodiments, the diagnostic methods disclosed herein, can beused to accurately and reliably detect ischemic injury of tissues, organtoxicity and kidney injury, including but not limited to acute kidneyinjury (AKI) and chronic kidney disease (CKD) in subjects, and subjects,(e.g., type I diabetic subjects) at risk of end stage renal disease(ESRD).

For example, in some embodiments where phospho-specific antibodies areused, e.g., phospho-specific antibodies to at least one of pT86, pS88,pT95, and phospho-specific antibodies to one or both of pT234, pS242,the presence of binding of a phospho-specific antibody to at least oneof pT86, pS88, pT95, and the absence of binding of a phospho-specificantibody to at least one of pT234, pS242 can be used to identify asubject with any one of: ischemia, organ toxicity, kidney injury, AKI orCKD.

In alternative embodiments, where phospho-specific antibodies are used,e.g., phospho-specific antibodies to at least one of pT86, pS88, pT95,and antibodies that specifically bind to one or both of nT234, nS242,the presence of binding of a phospho-specific antibody to at least oneof, or at least 2 of pT86, pS88, pT95, and the presence of binding of aspecific antibody to at least one of nT234, nS242 can be used toidentify a subject with any one of: ischemia, organ toxicity, kidneyinjury, AKI or CKD.

In some embodiments, where the phosphorylation status of one, or 2 or 3or 4 or 5 of pT86, pS88, pT95, nT234, nS242 of the NPM polypeptide aredetected, in particular, at least one of pT86, pS88, pT95 is detectedand at least one of nT234, nS242 of the NPM polypeptide are detected,the subject from whom the biological sample was obtained can be treatedwith an appropriate treatment for kidney injury or AKI as disclosedherein.

C. Methods and Assays to Detect Phosphorylation States of any of the 5Ischemic-Induced Phosphorylation/Dephosphorylation States of NPMPolypeptide (i.e., Methods to Detect pT86, pS88, PT95, nT234, nS242 onNPM Polypeptide).

In some embodiments, an agent which specifically binds to any one ormore of amino acid residues: pT86, pS88, pT95, pT234 or pS242 on the NPMpolypeptide is an antibody or antibody fragment (e.g., anantigen-binding Ab fragment), or a protein-binding molecule. Suitableantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, humanized, recombinant, single chain, F_(ab), F_(ab′), F_(sc),R_(v), and F_((ab′)2) fragments, and an F_(a)b expression library.Antibodies are readily raised in animals such as rabbits or mice byimmunization with the antigen. Immunized mice are particularly usefulfor providing sources of B cells for the manufacture of hybridomas,which in turn are cultured to produce large quantities of monoclonalantibodies. In general, an antibody molecule obtained from humans can beclassified in one of the immunoglobulin classes IgG, IgM, IgA, IgE andIgD, which differ from one another by the nature of the heavy chainpresent in the molecule. Certain classes have subclasses as well, suchas IgG₁, IgG₂, and others. Furthermore, in humans, the light chain maybe a kappa chain or a lambda chain. Reference herein to antibodiesincludes a reference to all such classes, subclasses and types of humanantibody species.

Antibodies provide high binding avidity and unique specificity to a widerange of target antigens and haptens. Monoclonal antibodies useful inthe practice of the methods disclosed herein include whole antibody andfragments thereof and are generated in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis.

The ischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242), or a portion or fragment thereof, can serve as anantigen, and additionally can be used as an immunogen to generateantibodies that immunospecifically bind the antigen, using standardtechniques for polyclonal and monoclonal antibody preparation.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, or at least 15 amino acid residues, or at least 20 amino acidresidues, or at least 30 amino acid residues.

Useful monoclonal antibodies and fragments can be derived from anyspecies (including humans) or can be formed as chimeric proteins whichemploy sequences from more than one species. Human monoclonal antibodiesor “humanized” murine antibody are also used in accordance with thepresent invention. For example, murine monoclonal antibody can be“humanized” by genetically recombining the nucleotide sequence encodingthe murine Fv region (i.e., containing the antigen binding sites) or thecomplementarily determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region. Humanizedtargeting moieties are recognized to decrease the immunoreactivity ofthe antibody or polypeptide in the host recipient, permitting anincrease in the half-life and a reduction the possibly of adverse immunereactions in a manner similar to that disclosed in European PatentApplication No. 0,411,893 A2. The murine monoclonal antibodies shouldpreferably be employed in humanized form. Antigen binding activity isdetermined by the sequences and conformation of the amino acids of thesix complementarily determining regions (CDRs) that are located (threeeach) on the light and heavy chains of the variable portion (Fv) of theantibody. The 25-kDa single-chain Fv (scFv) molecule, composed of avariable region (VL) of the light chain and a variable region (VH) ofthe heavy chain joined via a short peptide spacer sequence, is thesmallest antibody fragment developed to date. Techniques have beendeveloped to display scFv molecules on the surface of filamentous phagethat contain the gene for the scFv. scFv molecules with a broad range ofantigenic-specificities can be present in a single large pool ofscFv-phage library. Some examples of high affinity monoclonal antibodiesand chimeric derivatives thereof, useful in the methods of the presentinvention, are described in the European Patent Application EP 186,833;PCT Patent Application WO 92/16553; and U.S. Pat. No. 6,090,923.

Chimeric antibodies are immunoglobin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobin constant region is derived from a human immunoglobinmolecule. Preferably, both regions and the combination have lowimmunogenicity as routinely determined.

Anti-NPM antibodies are also commercially available from vendors such asBiorbyt (Cambridge, UK), GeneTex (Irvine, USA), Aviva Systems Biology(Beijing, CN), Bioss Inc. (Woburn, USA), Sino Biological (Beijing CN),Acris Antibodies GmbH (San Diego, USA), Raybiotech, Inc. (Norcross,USA), OriGene Technologies (Rockville, USA), Enzo Life Sciences(Farmingdale, USA), and Abeam (Cambridge, UK).

(i) Phospho-Specific NPM Antibodies:

Exemplary antibodies which can be used to identify an ischemic-inducedphosphorylated form of the NPM polypeptide (i.e., having thephosphorylation status of one or more of pT86, pS88, pT95, nT234, nS242)can be generated by one of ordinary skill in the art. In particular, onecan make antibodies or antibody fragments or antigen-binding fragmentsthereof that specifically bind to any of pT86, pS88, pT95, or pT234,pS242 according to the methods disclosed in U.S. Pat. No. 5,559,681,which is incorporated herein in its entirety by reference.

Generation of antibodies or antigen-binding fragments thereof thatspecifically bind to any of pT86, pS88, pT95, or pT234, pS242 (or nT234or nS242) of the NPM polypeptide can be generated usingactivation-specific phosphoprotein immunodetection (APHID). Inparticular, peptides of the ischemic-induced phosphorylated form of theNPM polypeptide (i.e., having the phosphorylation status of one or moreof pT86, pS88, pT95, nT234, nS242) can be generated, and coupled (via anamino terminal cysteine residue) to a carrier protein (e.g.,hemocyanin), combined with adjuvant and inoculated into rabbits.Following raising of polyclonal antisera, antibodies to thephosphorylated peptide can be purified in a reverse-purification processby adsorption of non-activation-specific antibodies to anunphosphorylated peptide of the same sequence. Where necessary,contaminating non-receptor-specific antiphosphotyrosine antibodies canbe removed by adsorption to phosphotyramine or phosphoserine.

APHID technology is generally applicable to the identification ofprotein isoforms characterized by varying phosphorylation states. Atypical scheme for preparing polyclonal antibodies foractivation-state-specific phosphoprotein immunodetection is shown inFIG. 1 of U.S. Pat. No. 5,559,681 which is incorporated herein in itsentirety by reference. A skilled artisan can use the APHID technology togenerate phospho-specific NPM polypeptides (e.g., antibodies thatspecifically bind NPM polypeptides that have one or more pT86, pS88,pT95 residues), or can detect dephosphorylation of specific residues ofNPM (e.g., antibodies that specifically bind to NPM polypeptides thathave one or both of nT234 or nS242).

(ii) Assays for Detection of Ischemic-Induced Phosphorylated Form of theNPM Polypeptide (i.e., Having the Phosphorylation Status of One or Moreof pT86, pS88, pT95, nT234, nS242).

RIA and ELISA provide the benefit of detection sensitivity, rapidity,accuracy, possible automation of procedures, and the like, for thedetermination of the presence of, or concentration or level of thephosphorylation state of any one of amino acid residue (i.e., T86, S88,T95, T234, S242) of the NPM polypeptide (Modern Rheumatology 13: 22-26(2003)), Ohkuni et al., (International Congress Series 1289: 71-74(2006)), and Mitchell et al., (Mol Microbiol. 5: 1883-8 (1991)).Radioimmunoassay (Kashyap, M. L. et al., J. Clin. Invest. 60:171-180(1977)) is a technique in which detection antibody can be used afterlabeling with a radioactive isotope such as 125I . Antibody arrays orprotein chips can also be employed, see for example U.S. PatentApplication Nos: 20030013208A1; 20020155493A1; 20030017515 and U.S. Pat.Nos. 6,329,209; 6,365,418, which are herein incorporated by reference intheir entirety.

Immunoassays

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA). There are different forms of ELISA which are well knownto those skilled in the art, e.g. standard ELISA, competitive ELISA, andsandwich ELISA. The standard techniques for ELISA are described in“Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. JohnWiley & Sons, 1980; Campbell et ah, “Methods and Immunology”, W. A.Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin.Biochem., 22:895-904. ELISA is a technique for detecting and measuringthe concentration of an antigen, such as an acute kidney injurybiomarker, using a labeled (e.g. enzyme linked) form of the antibody.

In a “sandwich ELISA”, an antibody is linked to a solid phase (i.e. amicrotiter plate) and exposed to a biological sample containing antigen(e.g. an acute kidney injury biomarker). The solid phase is then washedto remove unbound antigen. A labeled antibody (e.g. enzyme linked) isthen bound to the plate bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphatase, horseradish peroxidase,luciferase, urease, and B-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured.

In a “competitive ELISA”, a specific concentration of an antibodyspecific for the phosphorylation state of any one of amino acid residue(i.e., T86, S88, T95, T234, S242) of the NPM polypeptide is incubatedwith a biological sample. The antibody mixture is then contacted with asolid phase (e.g. a microtiter plate) that is coated with NPMpolypeptide. Where there the antibody binds to a phosphorylated residueon the NPM polypeptide, (e.g., any one of amino acid residue p T86,pS88, pT95, pT234, pS242), the less free antibody that will be availableto bind to the solid phase. A labeled (e.g., enzyme linked) secondaryantibody is then added to the solid phase to determine the amount ofprimary antibody bound to the solid phase.

In some embodiments, the phosphorylation states of one or more aminoacid residues of: T86, S88, T95, T234, S242 of the NPM polypeptide canbe determined simultaneously, in a multiplex fashion, by ELISA(enzyme-linked immunosorbent assay). The biological sample, e.g., urinesample can be, for example, one of a plurality of biological samplesobtained at one of the various timepoints from a subject in need. Insome embodiments, the biological sample is a human urine sample or bloodsample from a subject, to be tested for determining the phosphorylationstates of one or more residues T86, S88, T95, T234, S242 of the NPMpolypeptide according to the methods described herein. The urine orblood sample (e.g., plasma, serum etc.) from the individual may furtherbe serially diluted, according to the needs of the assay, and as knownto one of ordinary skill in the art. In some embodiments, one or more ofa plurality of antibodies or antigen-binding fragments specific for thephosphorylation state of each of T86, S88, T95, T234, S242 are assayedin a single sample which is contacted with the biological sample, thusforming an antibody-NPM complex or NPM-antigen-binding fragment complex.In some embodiments, each antibody or antigen-binding fragment specificfor each phosphorylated residue of T86, S88, T95, T234, S242 of NPMpolypeptide is labeled with a different label. In some embodiments, eachdifferent label is a fluorescent label. In all such embodiments, eachdifferent label has a unique emission spectra, such that each antibodycan be detected individually. Therefore, for example, one can determineif the individual antibodies or antigen-binding fragments bind to eachof the phosphorylated amino acid residues pT86, pS88, pT95, and wherethe individual antibodies or antigen-binding fragments do not bind tophosphorylated amino acid residues pT234 or pS242. Therefore, thephosphorylation state of each amino acid of T86, S88, T95, T234, S242can then be determined by calculating different or changes in theemission spectrum, wherein the relative intensity of signal from each ofthe fluorescent labels correlates with the binding of, and number ofantibodies against the particular amino acid residue (i.e., T86, S88,T95, T234, S242) being assayed. For example, if looking at only 2phosphorylation residues, T86 and S88, if pT86 is identified with a redfluorescent signal and pS88 is identified with a yellow signal, a redsignal would identify phosphorylation of just pT86, a yellow signalwould identify just phosphorylation of pS88, and an orange signal wouldidentify both pT86 and pS88 (which would identify that there is likelyAKI). In alternative embodiments, antibodies or antigen bindingfragments to detect the phosphorylation state of each amino acid residue(i.e., T86, S88, T95, T234, S242) can be assayed in a separate well. Thewells can be normalized to a well comprising all of the necessary ELISAreagents with the exception of the sample. A series of standards havingknown concentrations of each of the various biomarkers being assayedpermits actual quantification of the concentration of each of thebiomarkers in the sample.

In some aspects, the phosphorylation state of any one or more of aminoacid residues (i.e., T86, S88, T95, T234, S242) of the NPM polypeptidecan be assayed can be determined alone, or in combination with otherbiomarkers (e.g., other biomarkers for AKI such as for example, albuminor serum creatinine, KIM-1 or other disease pathologies, or anormalizing protein biomarker) simultaneously, in a multiplex fashion,using a multiplex bead assay. For example, in one embodiment, beads ofdifferent sizes or colors (emission spectra) are used for multiplexedimmunoassays to determine the phosphorylation state of each amino acidresidue (i.e., T86, S88, T95, T234, S242) of the NPM polypeptide andoptionally, one or more other blood biomarkers. In some embodiments ofthis aspect, a plurality of beads of different sizes are coated withdifferent antibodies, wherein each bead of a specific size is conjugatedto an antibody specific for a single biomarker (e.g., a bead of one sizeis conjugated to an antibody for pT86 and beads of different sizes areconjugated to different antibodies that are either specific to the otherphosphorylation residues (e.g., S88, T95, T234, S242) or specific todifferent blood biomarkers, or antibodies specific to normalizingproteins). Accordingly, each bead can be differentiated by its uniquelight scatter characteristics. A biological sample, such as a urine, orplasma or serum sample, to be assayed for the phosphorylation state ofat least one or more amino acid residue (i.e., T86, S88, T95, T234,S242) of the NPM polypeptide and optionally at least one other biomarkeris then contacted with a plurality of beads of different sizes, forminga bead-biomarker conjugate, and the phosphorylation state of each aminoacid residue (i.e., T86, S88, T95, T234, S242) of the NPM polypeptideand the concentration of other AKI biomarker can then be ascertained by,for example, performing flow cytometric analyses on the beadbound-sample. In some embodiments, one of the other biomarkers assessedin a multiplex bead assay is a normalizing protein to detect the levelof protein in the biological sample.

In some embodiments of this aspect, such bead-based technology can beemployed wherein bead populations are identified by one type offluorescence, while the biomarker-dependent signal is generated bydetection reagents carrying a second type of fluorescent signal, thuscreating a bead set specific for detecting the phosphorylation states ofeach amino acid residue (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide. In some embodiments, the bead-based technology can be usedto detect additional biomarkers, e.g., one or more other normalizingproteins, and/or other biomarkers (e.g., normalizing proteins and/ortotal NPM polypeptide or other AKI biomarker, e.g., KIM-1, albuiminuraand/or creatinine etc). In some embodiments, the distinguishable beadpopulations are prepared by staining the beads with two or morefluorescent dyes at various ratios. Each bead having a specific ratio ofthe two or more fluorescent dyes is conjugated to an antibody specificfor one of a plurality of biomarkers, thus assigning each bead a uniquefluorescent signature. The immunoassay signal is generated by detectionreagents, coupled to a third type of fluorescent dye. A sample to beassayed for the phosphorylation states of each amino acid residue (i.e.,T86, S88, T95, T234, S242) of the NPM polypeptide and optionally thetotal NPM polypeptide, is then contacted with the plurality of beadswith unique fluorescent signatures and specific detection of pT86, pS88,pT95, pT234, pS242, forming a bead-biomarker conjugate for NPM or otherbiomarker present in the sample. That way, the presence of signal forantibodies that specifically bind to pT86, pS88, pT95, and the absenceof a signal of antibodies that specifically bind to pT234 or pS242 canbe detected. Thus the phosphorylation states of each amino acid residue(i.e., T86, S88, T95, T234, S242) of the NPM polypeptide by the presenceor absence of a signal, as well as the concentration of the NPMpolypeptide can be ascertained by flow cytometric analyses on the beadbound-sample. For example, in some embodiments, beads are dyed withfluorochromes having different fluorescence intensities. In someembodiments, the beads are 7.5 μm in diameter. In some embodiments, thefluorescent dye incorporated in the beads fluoresces strongly at 650 nmupon excitation with an argon laser. Each bead population of a givenfluorescence intensity represents a discrete population for constructingan immunoassay for a single biomarker. Each bead population having agiven fluorescence intensity upon excitation is covalently coupled withan antibody directed against a specific biomarker, e.g., an antibody orantigen binding fragment that specifically binds to each of pT86, pS88,pT95, pT234, pS242. These antibody-bound bead populations, each of whichare unique in their fluorescence emission intensity, serve as capturebeads for a combination of detection of any one of the phosphorylationstates of each amino acid residue (i.e., T86, S88, T95, T234, S242) ofthe NPM polypeptide in a sample. That is, as stated above, thebiological sample can be detected with as many as 7 different beads;each bead having an antibody that is specific for one of pT86, pS88,pT95, pT234, pS242, total NPM polypeptide, and a control housekeepinggene. The presence of a signal from the beads that specifically detector bind to pT86, pS88, pT95, and the absence of the signal from thebeads that specifically detect or bind to pT234, pS242 can be used todetermine if the biological sample comprises the ischemic-inducedphospho-NPM polypeptide. Beads that specifically detect the total NPMpolypeptide and the housekeeping gene can serve as positive controlsand/or normalizing controls. In alternative embodiments, the beads cancomprise antibodies that specifically detect or bind to nT234 and/ornS242, where a presence of a signal from such beads can be used todetermine if the biological sample comprises the ischemic-inducedphospho-NPM polypeptide. It is envisioned that any combination ofantibodies can be used, that are specific for the phosphorylated pT86,pS88, pT95, pT234, pS242 residues on the NPM polypeptide, or arespecific for the non-phosphorylated residues (e.g., nT86, nS88, nT95,nT234, nS242).

Accordingly, as defined herein a “capture bead” is a bead having aunique fluorescence emission intensity conjugated to an antibodyspecific for the phosphorylation states of each amino acid residue(i.e., T86, S88, T95, T234, S242) of the NPM polypeptide. When thesecapture beads specific for different biomarkers are used as a mixture,the levels of individual biomarkers, the phosphorylation states of eachamino acid residue (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide, can be simultaneously measured within a given biologicalsample. In some embodiments, detection is further mediated by thebinding of a specific detection antibody, for example, an antibody thatdetects any bead-biomarker complex present in a sample, that is directlyconjugated with phycoerythrin (PE), to each of the corresponding capturebead-biomarker complexes present in the sample, thus providing a secondfluorescent signal for each capture bead. The fluorescent signal isproportional to the concentration of the biomarker in the sample.Separately established calibration curves can be used to determine theconcentration of each biomarker in the test sample, using dedicatedanalysis software, such as CBA software. The data collected using a flowcytometer include information about the physical and spectral parametersof the beads, such as size and the fluorescence emission characteristicsof each bead population. These fluorescence emission characteristicsinclude the fluorescent emission of the dyed beads, and the potentialfluorescent emissions of the detection fluorochrome (for example,phycoerythrin). When samples are analyzed using a flow cytometer inconjunction with a typical data acquisition and analysis package (fore.g., BD CellQuest™ software), a list-mode data file is saved using aflow cytometry standard file format, FCS. The data stored in the FCSfiles can be reanalyzed to determine the median fluorescence intensities(MFI) of the various bead populations, defined by their unique physicaland spectral characteristics, to then compare reference samples withunknowns. The level of the phosphorylation states of each amino acidresidue (i.e., T86, S88, T95, T234, S242) of the NPM polypeptide beingassayed within individual biological samples can then be calculated fromcalibration curves generated by serial dilutions of standard analytesolutions of known concentration. An automated or semiautomated analysismethod can be used for rapid reanalysis of the data stored in each FCSfile. For example, BD CBA Software is written in the Microsoft® ExcelVisual Basic for Applications (VBA) programming language. The CBASoftware can recognize FCS 2.0 and 3.0 format data files and automatesthe identification of CBA bead populations and the determination ofdetector fluorochrome MFI values for each bead population within thedata file for a single sample. Using this data analysis function of theCBA Software for multiple standard files, the MFI values for standardsare then determined and plotted. From the plotted standard curve andcomplex mathematical interpolation, values for unknown samples can berapidly determined in comparison to known standards using the software.

Other techniques can be used to detect the phosphorylation states ofeach amino acid residue (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide as required to practice the methods described herein,according to a practitioner's preference, and based upon the presentdisclosure. The suitability of a given method for measuring thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide will depend on the ability of thatmethod or assay to distinguish between each phosphorylation state, totalNPM protein levels and other proteins in the biological sample. Thus, animmunoassay can distinguish on the basis of selective binding to thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide and not another ser/thr/tyr(phosphorylated or non-phosphorylated) residue of the NPM polypeptide,or other protein in the biological sample. Spectrometric approaches canbe applied when a given agent will have a distinct spectrum or profilein the assay relative to others. One such technique is Western blotting(Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein asuitably treated sample is run on an SDS-PAGE gel before beingtransferred to a solid support, such as a nitrocellulose filter.Detectably labeled antibodies that specifically bind to thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide can then be used to detect thepresence or absence of phosphorylation at each site (e.g., amino acidresidues, T86, S88, T95, T234, S242), as well as the levels orconcentrations of NPM polypeptide, where the intensity of the signalfrom the detectable label corresponds to the amount of phosphorylationof each amino acid residue (i.e., T86, S88, T95, T234, S242) of the NPMprotein assessed as well as, in some embodiments, the amount of NPMprotein present. Levels can be quantitated, for example by densitometry.

The prognostic methods of the invention also are useful for determininga proper course of treatment for a patient having AKI. A course oftreatment refers to the therapeutic measures taken for a patient afterdiagnosis or after treatment for injury.

The present invention is also directed to commercial kits for thedetection and prognostic evaluation of AKI. The kit can be in anyconfiguration well known to those skilled in the art and is useful forperforming one or more of the methods described herein for the detectionof the phosphorylation states of each amino acid residue (i.e., T86,S88, T95, T234, S242) of the NPM polypeptide in a biological sampleobtained from a subject. The kits are convenient in that they supplymany, if not all, of the essential reagents for conducting an assay forthe detection of the phosphorylation states of each amino acid residue(i.e., T86, S88, T95, T234, S242) of the NPM polypeptide, such asdescribed herein. In addition, the assay may be performed simultaneouslywith a standard or multiple standards included in the kit, such as apredetermined amount of a NPM polypeptide, or positive or negativecontrols for detection of any one or more of pT86, pS88, pT95, pT234 (ornT234), pS242 (or nS242) so that the results of the test can bequantified or validated.

In one embodiment, the kit comprises a means for detecting thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide in a biological sample obtained fromthe subject. The kit can comprise a “dot blot” or a “dipstick” with atleast one antibody, antigen-binding fragment or binding agentimmobilized thereon, which specifically binds to one or more thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide. Specifically bound NPM protein canthen be detected using, for example, a second antibody that isdetectably labeled with a calorimetric agent or radioisotope.

In some embodiments, a kit comprises a paper-based assay to determinethe phosphorylation states of each amino acid residue (i.e., T86, S88,T95, T234, S242) of the NPM polypeptide in a biological sample. Suchpaper-based assays are well known in the art, e.g., as disclosed inInternational Application WO 2011097412 and U.S. Pat. No. 8,821,810 andUS application US 2014/0193840 and published documents by Martinez etal., (2007), Patterned Paper as a Platform for Inexpensive, Low-Volume,Portable Bioassays. Angewandte Chemie International Edition. 2007;46(8): 1318-1320, and Chung et al., (2010) Paper-Based ELISA. AngewandteChemie International Edition; 2010; 49(28):4771-4774, which are allincorporated herein in their entireties by reference.

In other embodiments, the assay kits may contain components forcompetitive and non-competitive assays, radioimmunoassay (RIA),multiplex bead assays, bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates, orimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity, and reproducibility of the assay areestablished by means well known to those skilled in the art.

In one embodiment, methods to detect the phosphorylation state of anyone or more of amino acid residues (i.e., T86, S88, T95, T234, S242) ofthe NPM polypeptide as disclosed herein include ELISA (enzyme linkedimmunosorbent assay), western blot, immunoprecipitation,immunofluorescence using detection reagents such as an antibody orprotein binding molecules or protein-binding agents. Alternatively, thephosphorylation states of each amino acid residue (i.e., T86, S88, T95,T234, S242) of the NPM polypeptide can be detected in a subject byintroducing into a subject a labeled antibody that specifically binds toat least one or more of the phosphorylation states of each amino acidresidue (i.e., T86, S88, T95, T234, S242) of the NPM polypeptide andother types of detection agent. For example, the antibody can be labeledwith a radioactive marker whose presence and location in the subject isdetected by standard imaging techniques, particularly useful are methodsthat detect the phosphorylation states of each amino acid residue (i.e.,T86, S88, T95, T234, S242) of the NPM polypeptide in a subject or in abiological sample.

Methods to detect the phosphorylation states of each amino acid residue(i.e., T86, S88, T95, T234, S242) of the NPM polypeptide in a biologicalsample are well known to persons skilled in the art, and are encompassedfor use in this invention. Commercially available antibodies and/orELISA kits for detection of one or more of the phosphorylation states ofeach amino acid residue (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide in a biological sample are also useful in the methods ofthis invention. Some examples of such protein-binding molecules usefulto detect the one or more phosphorylation states of each amino acidresidue (i.e., T86, S88, T95, T234, S242) of the NPM polypeptide arecommercially available, and include, but are not limited to,commercially available antibodies such as anti-phospho-Thr95 NPMantibody from AbCam, Bioss Antibodies, Cell Signaling Technologies (MA,USA), which can be found at world wide web site: “cellsignal-dot-com”.In some embodiments, antibodies from other antibody companies, such asfor example, Abnova corporation, Anogen, Alpco Diagnostics, Ray Biotech,alphagenix, autogen, R&D Systems, Pepro Tech EC Ltd, cytolab, BenderMedSystems GmbH, Biovision Research Products, EBD biosciences, Chemicon,Axxora Platform, Promo Cell Distrubuters, Cell Science, Santa CruzBiotechnology, Sigma etc. can be used. In alternative embodiments,antibodies directed against the ischemia-induced phospho-NPM polypeptidewith one or more phosphorylation states: T86, S88, T95, T234, S242 canalso be used in disease diagnostics and prognostics.

In another embodiment, immunohistochemistry (“IHC”) andimmunocytochemistry (“ICC”) techniques can be used. IHC is theapplication of immunochemistry to tissue sections, whereas ICC is theapplication of immunochemistry to cells or tissue imprints after theyhave undergone specific cytological preparations such as, for example,liquid-based preparations. Immunochemistry is a family of techniquesbased on the use of an antibody, wherein the antibodies are used tospecifically target molecules inside or on the surface of cells. Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change color, upon encountering thetargeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

In some embodiments, the methods as described herein can be performed,for example, by utilizing pre-packaged diagnostic kits, such as thosedescribed above, comprising at least one probe which can be convenientlyused, e.g., to determine whether a subject has or is at risk ofdeveloping disease such as AKI, CKD, ESRD and/or renal cell carcinoma(RCC), in particular clear cell renal cell carcinoma.

The term “protein-binding molecule” or “antibody-based binding moiety”or “antibody” includes immunoglobulin molecules and immunologicallyactive determinants of immunoglobulin molecules, e.g., molecules thatcontain an antigen binding site which specifically binds (i.e.immunoreacts with) to the Psap proteins. The term “antibody-basedbinding moiety” is intended to include whole antibodies, e.g., of anyisotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof whichare also specifically reactive with the Psap proteins. Antibodies can befragmented using conventional techniques. Thus, the term includessegments of proteolytically-cleaved or recombinantly-prepared portionsof an antibody molecule that are capable of selectively reacting with acertain protein. Non limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, dAbs and singlechain antibodies (scFv) containing a VL and VH domain joined by apeptide linker. The scFv's can be covalently or non-covalently linked toform antibodies having two or more binding sites. Thus, “antibody-basedbinding moiety” includes polyclonal, monoclonal, or other purifiedpreparations of antibodies and recombinant antibodies. The term“antibody-base binding moiety” is further intended to include humanizedantibodies, bispecific antibodies, and chimeric molecules having atleast one antigen binding determinant derived from an antibody molecule.In a preferred embodiment, the antibody-based binding moiety detectablylabeled.

The term “labeled antibody”, as used herein, includes antibodies thatare labeled by a detectable means and include, but are not limited to,antibodies that are enzymatically, radioactively, fluorescently, andchemiluminescently labeled. Antibodies can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. Thedetection and quantification of Psap or Tsp-1 present in the tissuesamples correlate to the intensity of the signal emitted from thedetectably labeled antibody.

In one embodiment, the antibody-based binding moiety is detectablylabeled by linking the antibody to an enzyme. The enzyme, in turn, whenexposed to it's substrate, will react with the substrate in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used to detectably label the antibodies of the presentinvention include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling an antibody, it ispossible to detect the antibody through the use of radioimmune assays.The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by audioradiography.Isotopes which are particularly useful for the purpose of the presentinvention are ³H, ¹³¹I, ³⁵S, ¹⁴C, and preferably ¹²⁵I.

It is also possible to label an antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are CYE dyes, fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

An antibody can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

An antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-antibodyis then determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of particularlyuseful chemiluminescent labeling compounds are luminol, luciferin,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

As mentioned above, levels of enzyme protein can be detected byimmunoassays, such as enzyme linked immunoabsorbent assay (ELISA),radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Westernblotting, immunocytochemistry or immunohistochemistry, each of which aredescribed in more detail below. Immunoassays such as ELISA or RIA, whichcan be extremely rapid, are more generally preferred. Antibody arrays orprotein chips can also be employed, see for example U.S. PatentApplication Nos: 20030013208A1; 20020155493A1; 20030017515 and U.S. Pat.Nos. 6,329,209; 6,365,418, which are herein incorporated by reference intheir entirety.

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA).” ELISA is a technique for detecting and measuring theconcentration of an antigen using a labeled (e.g. enzyme linked) form ofthe antibody. There are different forms of ELISA, which are well knownto those skilled in the art. The standard techniques known in the artfor ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition,Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al.,“Methods and Immunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M.1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

In a “sandwich ELISA”, an antibody (e.g. anti-enzyme) is linked to asolid phase (i.e. a microtiter plate) and exposed to a biological samplecontaining antigen (e.g. enzyme). The solid phase is then washed toremove unbound antigen. A labeled antibody (e.g. enzyme linked) is thenbound to the bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphatase, horseradish peroxidase,luciferase, urease, and B-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured.

In a “competitive ELISA”, antibody is incubated with a sample containingantigen (i.e. enzyme). The antigen-antibody mixture is then contactedwith a solid phase (e.g. a microtiter plate) that is coated with antigen(i.e., enzyme). The more antigen present in the sample, the less freeantibody that will be available to bind to the solid phase. A labeled(e.g., enzyme linked) secondary antibody is then added to the solidphase to determine the amount of primary antibody bound to the solidphase.

In an “immunohistochemistry assay” a section of tissue is tested forspecific proteins by exposing the tissue to antibodies that are specificfor the protein that is being assayed. The antibodies are thenvisualized by any of a number of methods to determine the presence andamount of the protein present. Examples of methods used to visualizeantibodies are, for example, through enzymes linked to the antibodies(e.g., luciferase, alkaline phosphatase, horseradish peroxidase, orbeta-galactosidase), or chemical methods (e.g., DAB/Substratechromagen). The sample is then analysed microscopically, most preferablyby light microscopy of a sample stained with a stain that is detected inthe visible spectrum, using any of a variety of such staining methodsand reagents known to those skilled in the art.

Alternatively, “Radioimmunoassays” can be employed. A radioimmunoassayis a technique for detecting and measuring the concentration of anantigen using a labeled (e.g. radioactively or fluorescently labeled)form of the antigen. Examples of radioactive labels for antigens include3H, 14C, and 125I. The concentration of antigen enzyme in a biologicalsample is measured by having the antigen in the biological samplecompete with the labeled (e.g. radioactively) antigen for binding to anantibody to the antigen. To ensure competitive binding between thelabeled antigen and the unlabeled antigen, the labeled antigen ispresent in a concentration sufficient to saturate the binding sites ofthe antibody. The higher the concentration of antigen in the sample, thelower the concentration of labeled antigen that will bind to theantibody.

In a radioimmunoassay, to determine the concentration of labeled antigenbound to antibody, the antigen-antibody complex must be separated fromthe free antigen. One method for separating the antigen-antibody complexfrom the free antigen is by precipitating the antigen-antibody complexwith an anti-isotype antiserum. Another method for separating theantigen-antibody complex from the free antigen is by precipitating theantigen-antibody complex with formalin-killed S. aureus. Yet anothermethod for separating the antigen-antibody complex from the free antigenis by performing a “solid-phase radioimmunoassay” where the antibody islinked (e.g., covalently) to Sepharose beads, polystyrene wells,polyvinylchloride wells, or microtiter wells. By comparing theconcentration of labeled antigen bound to antibody to a standard curvebased on samples having a known concentration of antigen, theconcentration of antigen in the biological sample can be determined.

An “immunoradiometric assay” (IRMA) is an immunoassay in which theantibody reagent is radioactively labeled. An IRMA requires theproduction of a multivalent antigen conjugate, by techniques such asconjugation to a protein e.g., rabbit serum albumin (RSA). Themultivalent antigen conjugate must have at least 2 antigen residues permolecule and the antigen residues must be of sufficient distance apartto allow binding by at least two antibodies to the antigen. For example,in an IRMA the multivalent antigen conjugate can be attached to a solidsurface such as a plastic sphere. Unlabeled “sample” antigen andantibody to antigen which is radioactively labeled are added to a testtube containing the multivalent antigen conjugate coated sphere. Theantigen in the sample competes with the multivalent antigen conjugatefor antigen antibody binding sites. After an appropriate incubationperiod, the unbound reactants are removed by washing and the amount ofradioactivity on the solid phase is determined. The amount of boundradioactive antibody is inversely proportional to the concentration ofantigen in the sample.

Other techniques can be used to detect the phosphorylation state of anyone or more of amino acid residues (i.e., T86, S88, T95, T234, S242) ofthe NPM polypeptide in a biological sample according to a practitioner'spreference, and based upon the present disclosure and the type ofbiological sample (i.e. plasma, urine, tissue sample etc). One suchtechnique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Detectably labeled anti-enzyme antibodies canthen be used to assess enzyme levels, where the intensity of the signalfrom the detectable label corresponds to the amount of enzyme present.Levels can be quantified, for example by densitometry.

Immunological methods are particularly useful in the methods asdisclosed herein, because they require only small quantities ofbiological material, and are easily performed and at multiple differentlocations. In some embodiments, such an immunological method useful inthe methods as disclosed herein uses a “lab-on-a-chip” device, involvinga single device to run a single or multiple biological samples andrequires minimal reagents and apparatus and is easily performed, makingthe “lab-on-a-chip” devices which detect the phosphorylation status, inparticular the phosphorylation status of S10 residue of a topo I proteinis ideal for rapid, on-site diagnostic tests to identify if a biologicalsample obtained from a subject is likely to be responsive to a topo Iinhibitor. In some embodiments, the immunological methods can be done atthe cellular level and thereby necessitate a minimum of one cell.Preferably, several cells are obtained from a subject affected with orat risk for developing cancer and assayed using tire methods, kits,machines, computer systems and media as disclosed herein.

Mass Spectrometry

In other embodiments, the phosphorylation state of any one or more ofamino acid residues (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide present in a biological sample (e.g., urine, kidney biopsywhole blood, plasma or serum etc.) can be determined by massspectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquidchromatography-mass spectrometry (LC-MS), gas chromatography-massspectrometry (GC-MS), high performance liquid chromatography-massspectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry,nuclear magnetic resonance spectrometry, or tandem mass spectrometry(e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. PatentApplication Nos: 2003/0199001, 2003/0134304, 2003/0077616, which areherein incorporated by reference in their entirety.

The terms “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., “Proteinchip surface enhanced laser desorption/ionization(SELDI) mass spectrometry: a novel protein biochip technology fordetection of prostate cancer biomarkers in complex protein mixtures,”Prostate Cancer and Prostatic Diseases 2: 264-76 (1999); and Merchantand Weinberger, “Recent advancements in surface-enhanced laserdesorption/ionization-time of flight-mass spectrometry,” Electrophoresis21: 1164-67 (2000), each of which is hereby incorporated by reference inits entirety, including all tables, figures, and claims. Massspectrometry methods are well known in the art and have been used toquantify and/or identify biomolecules, such as proteins and hormones(see, e.g., Li et al., (2000), Tibtech. 18:151-160; Starcevic et. al.,(2003), J. Chromatography B, 792: 197-204; Kushnir M M et. al. (2006),Clin. Chem. 52:120-128; Rowley et al. (2000), Methods 20: 383-397; andKuster and Mann (1998), Curr. Opin. Structural Biol. 8: 393-400).Further, mass spectrometric techniques have been developed that permitat least partial de novo sequencing of isolated proteins. Chait et al.,(1993), Science, 262:89-92; Keough et al., (1999), Proc. Natl. Acad.Sci. USA. 96:7131-6; reviewed in Bergman (2000), EXS 88:133-44. Variousmethods of ionization are known in the art. For examples, AtmosphericPressure Chemical Ionisation (APCI) Chemical Ionisation (CI) ElectronImpact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB)Field Desorption/Field Ionisation (FD/FI) Matrix Assisted FaserDesorption Ionisation (MAFDI) and Thermospray Ionisation (TSP) Incertain embodiments, a gas phase ion spectrophotometer is used. In otherembodiments, laser-desorption/ionization mass spectrometry is used toanalyze the sample. Modern laser desorption/ionization mass spectrometry(“FDI-MS”) can be practiced in two main variations: matrix assistedlaser desorption/ionization (“MAFDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MAFDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Faser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937(Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait). InSELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe biomarker of interest. In another variant, the surface isderivatized with energy absorbing molecules that are not desorbed whenstruck with the laser. In another variant, the surface is derivatizedwith molecules that bind the protein of interest and that contain aphotolytic bond that is broken upon application of the laser. In each ofthese methods, the derivatizing agent generally is localized to aspecific location on the substrate surface where the sample is applied.See, e.g., U.S. Pat. No. 5,719,060 and WO 98/59361. The two methods canbe combined by, for example, using a SELDI affinity surface to capturean analyte and adding matrix-containing liquid to the captured analyteto provide the energy absorbing material. For additional informationregarding mass spectrometers, see, e.g., Principles of InstrumentalAnalysis, 3rd edition., Skoog, Saunders College Publishing,Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology,4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094.Detection and quantification of the biomarker will typically depend onthe detection of signal intensity. For example, in certain embodiments,the signal strength of peak values from spectra of a first sample and asecond sample can be compared (e.g., visually, by computer analysisetc.), to determine the relative amounts of particular biomarker.Software programs such as the Biomarker Wizard program (CiphergenBiosystems, Inc., Fremont, Calif.) can be used to aid in analyzing massspectra. The mass spectrometers and their techniques are well known tothose of skill in the art. The various assays are described herein interms of the detection of the phosphorylation state of any one or moreof amino acid residues (i.e., T86, S88, T95, T234, S242) of the NPMpolypeptide in a biological sample, e.g., urine sample. It is understoodthat the assays can be readily adapted to detect other analytes asneeded e.g., for various other embodiments and or to detect proteinlevels and depending on the sample type, such as biopsy sample, wholeblood, plasma or serum.

Mass spectrometry methods are well known in the art and have been usedto quantify and/or identify biomolecules, such as proteins (see, e.g.,Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20:383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8:393-400). Further, mass spectrometric techniques have been developedthat permit at least partial de novo sequencing of isolated proteins.Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad.Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).

In certain embodiments, a gas phase ion spectrophotometer is used. Inother embodiments, laser-desorption/ionization mass spectrometry is usedto analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937(Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

In SELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe protein of interest. In another variant, the surface is derivatizedwith energy absorbing molecules that are not desorbed when struck withthe laser. In another variant, the surface is derivatized with moleculesthat bind the protein of interest and that contain a photolytic bondthat is broken upon application of the laser. In each of these methods,the derivatizing agent generally is localized to a specific location onthe substrate surface where the sample is applied. See, e.g., U.S. Pat.No. 5,719,060 and WO 98/59361. The two methods can be combined by, forexample, using a SELDI affinity surface to capture an analyte and addingmatrix-containing liquid to the captured analyte to provide the energyabsorbing material.

For additional information regarding mass spectrometers, see, e.g.,Principles of Instrumental Analysis, 3rd edition., Skoog, SaundersCollege Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia ofChemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York1995), pp. 1071-1094.

Detection of the phosphorylation state of any one or more of amino acidresidues (i.e., T86, S88, T95, T234, S242) of the NPM polypeptide in abiological sample will typically depend on the detection of signalintensity. This, in turn, can reflect the quantity and character of apolypeptide bound to the substrate. For example, in certain embodiments,the signal strength of peak values from spectra of a first sample and asecond sample can be compared (e.g., visually, by computer analysisetc.), to determine the relative amounts of particular biomolecules.Software programs such as the Biomarker Wizard program (CiphergenBiosystems, Inc., Fremont, Calif.) can be used to aid in analyzing massspectra. The mass spectrometers and their techniques are well known tothose of skill in the art.

D. Biological Samples

In some embodiments, the detection of one or more phosphorylationsstates of amino acid residues T86, S88, T95, T234, S242 of the NPMpolypeptide is assessed in a biological sample obtained from a subject.In some embodiments, a biological sample is a sample of biologicalfluid, tissue, or cells, in a healthy and/or pathological state obtainedfrom a subject. Such samples include, but are not limited to, urine,whole blood, serum, plasma, sputum, saliva, amniotic fluid, lymph fluid,tissue or fine needle biopsy samples (including kidney biopsy samples),peritoneal fluid, cerebrospinal fluid, nipple aspirates, and includessupernatant from cell lysates, lysed cells, cellular extracts, andnuclear extracts. In some embodiments, the whole blood sample is furtherprocessed into serum or plasma samples. In some embodiments, a sample istaken from a human subject, e.g., a subject selected for diagnosis asdiscussed in section IV.E herein.

Accordingly, in one embodiment of this aspect and all other aspectsdescribed herein, a biological sample as defined herein can include ahuman biological sample, preferably a urine sample or a microdissectedhuman samples, are derived from a small tissue fraction, particularlyfrom a kidney tissue fraction. In some embodiments, the human samplesare preferably harvested by biopsy and/or surgical extraction, and insome embodiments, the human sample can be stored, for example as frozenbiological sample prior to subjecting to the detection ofphosphorylation status of the NPM polypeptide using the methods, kits,and media as disclosed herein.

In some embodiments of these methods and all such methods describedherein, the biological sample is a kidney biopsy, urine, blood, serumsample, or cells pelleted from a urine sample.

In some embodiments, the biological sample is treated after it isobtained from a subject to “fix” the phosphorylation status of NPMpolypeptide, such that there is not a change in phosphorylation status(i.e. a dephosphorylation or increase in phosphorylation) of NPMpolypeptide from the time the tissue (i.e. biological sample) washarvested from the subject and the time it is analysed by the methodsand kits as disclosed herein. In some embodiments, this is important,because phosphorylation status can rapidly alter (i.e. dephosphoryle orphosphorylate) if the phosphorylation status is not stable after removalof a biological sample or tissue from a subject, thus dephosphorylationmay increase or phosphorylation may increase leading to inaccuracies(i.e. false positives or false negatives) in the detection and analysisas disclosed herein. Accordingly, in some embodiments, a biologicalsample, such as urine or tissue biopsy is optionally treated to “fix”the phosphorylation status of topol polypeptide before the biologicalsample is subject to analysis by the methods, kits, machines, computersystems and computer readable media as disclosed herein. Methods to fixa biological sample, such as a tissue biopsy are well known by a skilledartisan, and include formaldehyde, formalin, FAA fixative and otherfixatives and methods commonly known by a skilled artisan.

E. Subjects Amenable for Diagnosis of Kidney Injury or Treatment withNPM Peptides

In all aspects of the invention, the assays, kits and methods, e.g.,methods for treating acute stress, ischemia or kidney injury, includingAKI in a subject, or diagnosis methods disclosed herein, e.g., methodsfor monitoring the effectiveness of a treatment (e.g., method to monitortreatment progress) is performed on a subject who is suspected to have,or has been previously diagnosed with or identified as suffering from orhaving a kidney injury, e.g., AKI, or injury to the proximal tubule ofthe kidney. In some embodiments, the diagnosis methods are performed ona biological sample obtained from a subject who has been selected to bediagnosed with, or suspected to have a condition in need of treatment(e.g. acute kidney injury, chronic kidney disease, end-stage renaldisease, or diabetic nephropathy) or one or more complications relatedto such a condition, or optionally, having undergone, or to undergo acardiopulmonary bypass (CBP), or have already undergone treatment forsuch a condition. The subject can also have been selected as being riskof developing a condition associated with kidney fibrosis. For example,acute kidney injury is now appreciated to be significantly associatedwith increased risk of future chronic kidney disease and end-stage renaldisease.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) used in the assays, methods and kits as disclosed herein is froma subject selected to be assessed, where the subject has been identifiedto suffer from an insult or injury to the kidney, e.g., an injury to theproximal tube of the kidney.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) used in the assays, methods and kits as disclosed herein is froma subject selected to be assessed, where the subject has been identifiedto suffer from acute kidney injury, and the methods described herein areused to treat the subject from developing chronic kidney disease. Insome embodiments, the method as used herein are used to prevent theworsening of a symptom of AKI, and/or monitoring the progression of AKI.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) used in the assays, methods and kits as disclosed herein is froma subject selected to be assessed, where the subject has been identifiedto suffer from chronic kidney disease or after a cardiopulmonary bypass(CBP) operation, and the methods described herein are used to preventthe subject from progressing to end-stage renal disease.

Common symptoms of chronic kidney disease include tiredness, nausea,urine-like odor to the breath, bone pain, abnormally dark or light skin,itching, restless leg syndrome, blood in stools, bruising easily, pedaledema, and peripheral edema. Chronic kidney disease can be diagnosedthrough, e.g., medical history, a blood test that measures completeblood count, BUN level, or creatinine level, renal flow and scan, andrenal ultrasound.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) used in the assays, methods and kits as disclosed herein is froma subject selected to be assessed, where the subject has been identifiedto have diabetes, e.g., type 1 diabetes. In some embodiments, themethods described herein are used to monitor the kidney injury in such asubject, and to monitor and optionally treat the subject prevent thesubject from progressing to end-stage renal disease.

When the kidneys are clearly beginning to shut down, it is called endstage renal disease. Symptoms of end-stage renal disease include, butare not limited to, a decrease in urine output, inability to urinate,fatigue, headaches, unexplained weight loss, loss of appetite, nauseaand vomiting, dry skin and itching, changes in skin color, bone pain,confusion and difficulty concentrating, bruising easily, numbness inhands and feet, bad breath, excessive thirst, and frequent hiccups.End-stage renal disease can be diagnosed through, e.g., a physicalexamination and blood tests to check kidney function.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) used in the assays, methods and kits as disclosed herein is froma subject selected to be assessed, where the subject has been identifiedto have diabetic neuropathy. Symptoms of diabetic nephropathy include,but are not limited to, lomerular hypertrophy, basement membranethickening, and mesgangial expansion. Diabetic nephropathy can bediagnosed and/or monitored using blood or urine tests, as well as bykidney biopsy. Such tests may be used to monitor improvement of symptomsduring or following treatment. By way of example, diabetic nephropathycan be diagnosed and/or assessed by evaluating blood and/or proteincontent in the urine. Diabetic nephropathy can also be diagnosed and/orassessed by evaluating creatinine and/or urea level in blood, and/or byestimates of glomerular filtration rate based on creatinine score.

In some embodiments, the biological sample (e.g., urine sample or bloodsample) in the assays, methods and kits as disclosed herein is from asubject selected to be assessed, where the subject has been identifiedto have kidney fibrosis or is at risk of developing kidney fibrosis. Forexample, the risk of developing kidney fibrosis is increased if thekidney suffers from an injury or insult. A condition associated withkidney fibrosis can be diagnosed by a blood test that measures the levelof waste products such as creatinine and urea, a urine test that looksfor abnormalities, an imaging test using ultrasound to assess kidney'sstructure and size, or a kidney biopsy.

F. Appropriate Therapy for the Treatment of Kidney Injury or KidneyDisease or AKI

In some embodiments, the methods and assays further comprise providingan appropriate treatment to the subject for kidney injury, e.g., wherethe biological sample obtained from subject is identified to have a NPMpolypeptide having the phosphorylation state of any one or more of aminoacids: pT86, pS88, pT95, nT234, nS242. The management of acute kidneyinjury hinges, in part, on identification and treatment of theunderlying cause. In addition to treatment of the underlying disorder,management of acute kidney injury can include the avoidance ofsubstances that are toxic to the kidneys, or “nephrotoxins,” whichinclude, but are not limited to, non-steroidal anti-inflammatory drugs(NSAIDs), such as ibuprofen, iodinated contrasts, such as those used forCT scans, and others. Therefore, in some embodiments, an appropriatetreatment for kidney injury or disease is to avoid nephrotoxins,including the subject reducing or stopping alcohol consumption andadministration of drugs e.g., NSAIDS and/or other non-necessarypharmaceutical compounds.

The choice of a specific therapeutic treatment for acute kidney injuryis dependent, in part, on the cause of the acute renal injury, i.e.,whether the cause of the acute kidney injury is pre-renal, renalinstrinsic, or post-renal. For example, in pre-renal acute kidney injuryin the absence of fluid overload, administration of intravenous fluidsis typically the first step to improve renal function. Fluidadministration may be monitored, for example, with the use of a centralvenous catheter to avoid over- or under-replacement of fluid. Insituations where low blood pressure is a persistent problem in the fluidreplete patient, inotropes, such as norepinephrine and dobutamine, maybe given to improve cardiac output and hence renal perfusion. In someembodiments, dopamine may be administered. In cases of prerenal acutekidney injury induced by toxins, discontinuation of the offending agent,such as aminoglycoside, penicillin, NSAIDs, or acetaminophen, can be aneffective treatment. If the cause of acute kidney injury is obstructionof the urinary tract, relief of the obstruction (with a nephrostomy orurinary catheter) may be necessary.

In cases where the acute kidney injury has renal intrinsic causes,specific therapies and treatment regimens are administered based on thenature of the renal intrinsic cause. For example, intrinsic acute kidneyinjury due to Wegener's granulomatosis may respond to steroidmedication.

Renal replacement therapy, such as hemodialysis or continuous venovenoushemofiltration (CVVH), may be instituted in some cases of acute kidneyinjury. Metabolic acidosis and hyperkalemia, the two most seriousbiochemical manifestations of acute renal failure, may require medicaltreatment with sodium bicarbonate administration and antihyperkalemicmeasures, unless dialysis is required.

In some cases of acute kidney injury, lack of improvement aftertreatment with fluid resuscitation, therapy-resistant hyperkalemia,metabolic acidosis, or fluid overload may necessitate artificial supportin the form of dialysis or hemofiltration.

Accordingly, in some embodiments, an appropriate treatment for kidneyinjury or disease is any of, or a combination of; intravenous fluidadministration (e.g. fluid resuscitation or fluid overload),hyperkalemia, metabolic acidosis, administration of inotropes (e.g.,norepinephrine and dobutamine), administration of dopamine,discontinuation of an offending agent, e.g., aminoglycoside, penicillin,NSAIDs, or acetaminophen and/or alcohol, dialysis, administration ofsteroids, hemodialysis or continuous venovenous hemofiltration (CVVH),administration of sodium bicarbonate administration, and/orantihyperkalemic measures.

In some cases of acute kidney injury, in which end-stage renal failurehas occurred, an appropriate treatment involves a kidney transplant. Asdefined herein, a “kidney transplant” or “renal transplant” is the organtransplant of a kidney into a patient with end-stage renal disease.Kidney transplantation is typically classified as deceased-donor(formerly known as cadaveric) or living-donor transplantation dependingon the source of the recipient organ. Living-donor renal transplants arefurther characterized as genetically related (living-related) ornon-related (living-unrelated) transplants, depending on whether abiological relationship exists between the donor and recipient.

In some embodiments, an appropriate treatment for kidney injury cancomprise administration of a treatment to the subject, e.g., alone or aspart of a combinatorial therapy. For example, TGF-β inhibitors can beadministered to hamper the progression of kidney fibrosis. Non-limitingexamples of agents and/or therapies which can be used to treat chronickidney disease, end-stage renal disease, or diabetic nephropathy includeany, or any combination of angiotensin converting enzyme inhibitors(ACEIs), angiotensin II receptor antagonists (ARBs), bardoxolone methyl,olmesartan medoxomil, sulodexide, avosentan, and renal replacementtherapy.

The efficacy of a given treatment for acute kidney injury can bedetermined by the skilled clinician, for example, using the criteriadiscussed herein. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of acute kidney injury, such as in one example, urinecreatinine levels, are altered in a beneficial manner, other clinicallyaccepted symptoms or markers of disease are improved, or evenameliorated, e.g., by at least 10% following treatment. Efficacy canalso be measured by a failure of an individual to worsen as assessed byhospitalization or need for medical interventions (i.e., progression ofthe disease is halted or at least slowed). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein. Treatment includes any treatment of a acute kidney injurydisease in an individual or an animal (some non-limiting examplesinclude a human, or a mammal) and includes: (1) inhibiting the disease,e.g., arresting, or slowing the progression of acute kidney injury oracute kidney injury complications; or (2) relieving the disease, e.g.,causing regression of symptoms, e.g., normalizing or reducing urinecreatinine levels; and (3) preventing or reducing the likelihood of thedevelopment of a further acute kidney injury complication, or the needfor administration of a further treatment, such as for example, a renaltransplant.

In some embodiments of these methods and all such methods describedherein, the method further comprises administering to the subject anadditional therapeutic agent, in addition to the inhibitor of Bax-NPMcomplex formation or NPM peptide as described herein. Such an additionaltherapeutic agent can be co-administered with the inhibitor of Bax-NPMcomplex formation or NPM peptide. As used herein, the phrase“co-administering” or to “co-administer” means the administration of aninhibitor of Bax-NPM complex formation or NPM peptide inhibitordescribed herein and another compound, e.g., a therapeutic agent,separately, simultaneously, and/or sequentially over a period of time asdetermined by a qualified care giver.

In some such embodiments, the additional therapeutic agent is anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin IIreceptor blocker (ARB), or a mineralocorticoid receptor (MR) antagonist.

ACE inhibitors for use with an inhibitor of Bax-NPM complex formation orNPM peptide described herein include, but are not limited to, benazepril(marketed in the U.S. as LOTENSIN™), captopril (marketed in the U.S. asCAPOTEN™), enalapril/enalaprilat (marketed in the U.S. as VASOTEC™ oraland injectable), fosinopril (marketed in the U.S. as MONOPRIL™),lisinopril (marketed in the U.S. as ZESTRIL™ and PRINIVIL™), moexipril(marketed in the U.S. as UNIVASC™), perindopril (marketed in the U.S. asACEON™), quinapril (marketed in the U.S. as ACCUPRIL™), ramipril(marketed in the U.S. as ALTACE™), and trandolapril (marketed in theU.S. as MAVIK™). ARBs for use with the ROBO2 inhibitors described hereininclude candesartan (marketed in the U.S. as ATACAND™), irbesartan(marketed in the U.S. as AVAPRO™), olmesartan (marketed in the U.S. asBENICAR™), losartan (marketed in the U.S. as COZAAR™), valsartan(marketed in the U.S. as DIOVAN™), telmisartan (marketed in the U.S. asMICARDIS™), and eprosartan (marketed in the U.S. as TEVETEN™).

In some embodiments of these methods and all such methods describedherein, the method further comprises administering to the subject aneffective amount of a diuretic, in addition to an inhibitor of Bax-NPMcomplex formation or NPM peptide. Diuretics include, but are not limitedto, torsemide (marketed in the U.S. as DEMADEX™), furosemide (marketedin the U.S. as LASIX™), bumetanide (marketed in the U.S. as BUMEX™),ethacrynic acid (marketed in the U.S. as EDECRIN™), torsemide (marketedin the U.S. as DEMADEX™), amiloride, (marketed in the U.S. as MIDAMOR™), acetazolamide (marketed in the U.S. as DIAMOX™), pamabrom(marketed in the U.S. as AQUA-BAN™), mannitol (marketed in the U.S. asARIDOL™ or OSMITROL™), traimterene (marketed in the U.S. as DYRENIUM™),spironolactone (marketed in the U.S. as ALDACTONE™), amiloride (marketedin the U.S. as MIDAMOR™), indapamide (marketed in the U.S. as LOZOL™),hydrochlorothiazide (marketed in the U.S. as HYDRODIURIL™), metolazone(marketed in the U.S. as ZAROXOLYN™ or MYKROX™), methylclothiazide(marketed in the U.S. as AQUATENSEN™ or ENDURON™), hydrocholorthiazide(marketed in the U.S. as AQUAZIDE H™ or ESIDRIX™ or MICROZIDE™),chlorothiazide (marketed in the U.S. as DIURIL™), bendroflumethiazide(marketed in the U.S. as NATURETIN™), polythiazide (marketed in the U.S.as RENESE™), hydroflumethiazide (marketed in the U.S. as SALURON™), andchlorthalidone (marketed in the U.S. as THALITONE™). For a completelisting also see, e.g., Physician's Desk Reference, 2012 Edition, PDRNetwork (2011).

An effective amount for the treatment of a disease means that amountwhich, when administered to a mammal in need thereof, is sufficient toresult in effective treatment as that term is defined herein, for thatdisease.

G. Determination of Stress-Induced Phosphorylation of NPM forDetermining Chemotherapy Efficacy and Toxicity.

In the research context, embodiments of the invention may provide amethod for drug screening and reporting of drug effects in preclinicaland clinical trials. The diagnostic methods disclosed herein can be usedto identify which subjects are being effectively treated with a cancertreatment or chemotherapy, assess the effectiveness of cancer treatmentor chemotherapy in a population of subjects, improve the quality andreduce costs of clinical trials, improve therapeutic success rates,and/or reduce sample sizes, trial duration and costs of clinical trials.

In particular, in one embodiment measuring NPM phosphorylation and celllocalization are useful to predict the severity and prognosis of acutetissue injury, and that NPM leakage into blood and/or urine represents anovel measure of acute cell death (a primary and desirable endpoint) incancer patients. In some embodiments, detection of NPM phosphorylationstates as well as presence of urinary NPM (and well as determining itsphosphorylated from) can be used for titration of chemo- and radiationtherapies. In some instances, detection of urinary NPM can be used todetermine if the subject should discontinue the nephrotoxic medications(including cancer treatments) or limit procedures in which renalischemia predictably occurs. In another instance, the leakage of NPM orphospho-NPM into the serum can be used as a biomarker of myocardialinfarction (MI) and/or ischemic stroke and can be used to determine theseverity and/or prognosis of each.

In some embodiments, the diagnostic assays and methods disclosed hereinfor detecting the different NPM phosphorylation states us useful totitrate chemotherapy and radiation to improve treatment efficacy. Insome embodiments, measuring urinary NPM leakage can be used to tract thesuitability of donated kidneys for transplantation. In some embodiments,detection of urinary NPM, including the phosphorylation states of theNPM polypeptide can be used to guide the discontinuation of nephrotoxinmedications (e.g., antibiotics, etc.) and predict tissue injury duringprocedures in which renal ischemia is predicted (e.g., bypassprocedures). In some embodiments, detection of NPM staining andphosphorylated forms of NPM polypeptide in renal biopsy samples isuseful for diagnosis and prognosis of acute tubular injury and otherforms of AKI (acute kidney injury). In some embodiments, detection ofNPM mutations in diverse cancer types can be used to predict sensitivityto chemotherapy and/or radiation.

Accordingly, one can use the diagnostic methods and kits disclosedherein for determining the effectiveness of cancer treatment and/oranticancer agents by determining the phosphorylation state of the NPMpolypeptide in urine from the subject, at any time before, during andafter anti-cancer treatments. If the cancer treatment appearsexcessively toxic, one can stop or decrease the cancer treatment.Alternatively, if the cancer treatment is not appearing effective, adifferent cancer treatment can be selected and/or the dose of theanti-cancer agent can be increased. Exemplary methods described in U.S.Pat. No. 7,344,829, which is incorporated herein in its entirety byreference, to titrate the cancer treatment based can be adapted to themethods disclosed herein based on the detection of the phosphorylationstate of the NPM polypeptide.

In the health care context, embodiments of the invention may provide aservice to physicians that will enable the physicians to tailor optimalpersonalized patient therapies. For example, a biological sample takenfrom a subject can be sent by the pathologist and/or clinical oncologistto a laboratory facility, for example, one such lab is operated byTheranostics Health, LLC. The laboratory may analyze the phosphorylationstates of one or more amino acid residues of: T86, S88, T95, T234, S242of the NPM polypeptide in the biological sample and provide a report tothe physician or health care provider. The laboratory may provide thetreating pathologist or clinical oncologist with a report indicating ifthe subject from which the biological sample was taken has, or is atrisk of having ischemic stress, kidney injury or AKI as disclosed hereinand optionally provide a recommendment on if the subject should betreated for kidney injury, ischemia etc. This may enable a physician totailor therapy to the individual subject, e.g., prescribe the righttherapy to the right patient at right time, provide a higher treatmentsuccess rate, spare the patient unnecessary toxicity and side effects,reduce the cost to patients and insurers of unnecessary or dangerousineffective medication, and improve patient quality of life, eventuallymaking cancer a managed disease, with follow up assays as appropriate.Physicians can use the reported information to tailor optimalpersonalized patient therapies instead of the current “trial and error”or “one size fits all” methods used to prescribe chemotherapy undercurrent systems. The inventive methods may establish a system ofpersonalized medicine.

Cancer Treatments

Several cancer therapies are known in the art and the use one or more ofthe various anticancer agents know will comprise in the context of thisinvention as a cancer treatment. An “anti-cancer” agent is capable ofnegatively affecting cancer in a subject, for example, by killing one ormore cancer cells, inducing apoptosis in one or more cancer cells,reducing the growth rate of one or more cancer cells, reducing theincidence or number of metastases, reducing a tumor's size, inhibiting atumor's growth, reducing the blood supply to a tumor or one or morecancer cells, promoting an immune response against one or more cancercells or a tumor, preventing or inhibiting the progression of a cancer,or increasing the lifespan of a subject with a cancer. Anti-canceragents include, for example, chemotherapy agents (chemotherapy),radiotherapy agents (radiotherapy), a surgical procedure (surgery),immune therapy agents (immunotherapy), genetic therapy agents (genetherapy), hormonal therapy, other biological agents (biotherapy) and/oralternative therapies.

A. Chemotherapeutic Agents

The term “chemotherapy” refers to the use of drugs to treat cancer. A“chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer. Some examples ofchemotherapeutic agents include antibiotic chemotherapeutics such as,Doxorubicin, Daunorubicin, Mitomycin (also known as mutamycin and/ormitomycin-C), Actinomycin D (Dactinomycin), Bleomycin, Plicomycin. Plantalkaloids such as Taxol, Vincristine, Vinblastine. Miscellaneous agentssuch as Cisplatin, VP 16, Tumor Necrosis Factor. Alkylating Agents suchas, Carmustine, Melphalan (also known as alkeran, L-phenylalaninemustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is aphenylalanine derivative of nitrogen mustard), Cyclophosphamide,Chlorambucil, Busulfan (also known as myleran), Lomustine. And otheragents for example, Cisplatin (CDDP), Carboplatin, Procarbazine,Mechlorethamine, Camptothecin, Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen Receptor Binding Agents,Gemcitabien, Navelbine, Famesyl-protein transferase inhibitors,Transplatinum, 5-Fluorouracil, and Methotrexate, Temazolomide (anaqueous form of DTIC), or any analog or derivative variant of theforegoing. Table 1 in U.S. Pat. No. 7,344,829, which is incorporatedherein in its entirety, lists numerous chemotherapeutics and their usein different cancer types.

Several chemotherapeutic agents change the phosphorylation state ofgrowth factor receptors in cancer cells. For example the protein kinaseinhibitor PKI166 decreases the amount of phosphorylated EGFR in cancercells, which is indicative of tumor shrinkage and decrease ofmetastasis. Thus, protein kinase inhibitor drugs are another major classof chemotherapeutic compounds important in the context of the presentinvention.

B. Radiotherapeutic Agents

Radiotherapeutic agents include radiation and waves that induce DNAdamage for example, γ-irradiation, X-rays, UV-irradiation, microwaves,electronic emissions, radioisotopes, and the like. Therapy may beachieved by irradiating the localized tumor site with the abovedescribed forms of radiations. It is most likely that all of theseagents effect a broad range of damage DNA, on the precursors of DNA, thereplication and repair of DNA, and the assembly and maintenance ofchromosomes.

Radiotherapeutic agents and methods of administration, dosages, etc.,are well known to those of skill in the art, and may be combined withthe invention in light of the disclosures herein. For example, dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

C. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes, for example, preventative, diagnostic or staging,curative and palliative surgery. Surgery, and in particular a curativesurgery, may be used in conjunction with other therapies, such as thepresent invention and one or more other agents.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised and/or destroyed. It is furthercontemplated that surgery may remove, excise or destroy superficialcancers, precancers, or incidental amounts of normal tissue. Treatmentby surgery includes for example, tumor resection, laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). Tumor resection refers to physical removal of at least part ofa tumor. Upon excision of part of all of cancerous cells, tissue, ortumor, a cavity may be formed in the body.

D. Immunotherapeutic Agents

An immunotherapeutic agent generally relies on the use of immuneeffector cells and molecules to target and destroy cancer cells. Theimmune effector may be, for example, an antibody specific for somemarker on the surface of a tumor cell. The antibody alone may serve asan effector of therapy or it may recruit other cells to actually effectcell killing. The antibody also may be conjugated to a drug or toxin(e.g., a chemotherapeutic, a radionuclide, a ricin A chain, a choleratoxin, a pertussis toxin, etc.) and serve merely as a targeting agent.Such antibody conjugates are called immunotoxins, and are well known inthe art (see U.S. Pat. Nos. 5,686,072, 5,578,706, 4,792,447, 5,045,451,4,664,911, and 5,767,072, each incorporated herein by reference).Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present invention. Common tumormarkers include carcinoembryonic antigen, prostate specific antigen,urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,laminin receptor, erb B and p155.

E. Genetic Therapy Agents

A tumor cell resistance to agents, such as chemotherapeutic andradiotherapeutic agents, represents a major problem in clinicaloncology. Improvement of the efficacy of one or more anti-cancer agentsis possible by combining such an agent with gene therapy. For example,the herpes simplex-thymidine kinase (HS-tK) gene, when delivered tobrain tumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver, et al.,1992). Gene therapy agents could encode proteins or antisense moleculesthat change NPM polypeptide phosphorylation, for example genes encodinggrowth factors. In the context of the present invention, it iscontemplated that gene therapy could be used similarly alone or inconjunction with other anticancer agents and one may determine theefficacy of such a treatment using methods of the present invention.

F. Combination Anticancer Therapies

A variety of cancer therapies such as those described above may be usedin context of the present invention. Generally, in order to increase theeffectiveness of the cancer therapy, it may be desirable to combine twoor more anticancer agents.

Administration of one anticancer agent may precede or follow the otheranticancer agent by intervals ranging from minutes to days to weeks. Inembodiments where both anticancer agents are administered together, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery. In such instances, it is contemplatedthat one would administer to a patient both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of either agentwill be required to achieve complete cancer cure. Various combinationsmay be employed, where the one anticancer agent is “A” and another is“B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations also are contemplated an assay of improvement orreduction of phosphorylation state of the NPM polypeptide as describedin the present invention can be performed at any stage during thesecombination treatments to determine the efficacy of the treatment and toreadjust the administration of drugs as required.

V. Kits

In further embodiments, the invention provides kits for use in detectingthe ischemic-induced phosphorylated form of the NPM polypeptide (i.e.,having the phosphorylation status of one or more of pT86, pS88, pT95,nT234, nS242, in biological samples obtained from a subject. Such kitswill generally comprise one or more antibodies that haveimmunospecificity for the phosphorylated NPM polypeptide as described inthe present invention.

The kits will thus comprise, in suitable container means, (a) componentsfor preserving the phosphorylation state of the NPM protein in abiological and (b) components for determining the phosphorylation statesof one or more amino acid residues of: T86, S88, T95, T234, S242 of theNPM polypeptide.

Kits comprising antibodies, such as site specific anti-phosphoantibodies that specifically bind to one or more of pT86, pS88, pT95,pT234, pS242 on the NPM polypeptide, or site specific antibodies whichspecifically binds to the nT234 or nS242 residues on the NPMpolypeptide, are envisioned. In some embodiments, it is contemplatedthat the antibodies will be those that bind to the ischemic-inducedphosphorylated form of the NPM polypeptide (i.e., having thephosphorylation status of one or more of pT86, pS88, pT95, nT234, nS242)in a biological sample. Monoclonal antibodies are readily prepared andwill often be preferred.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with, orlinked to, the given antibody or antigen itself. Detectable labels thatare associated with or attached to a secondary binding ligand are alsocontemplated. Exemplary secondary ligands are those secondary antibodiesthat have binding affinity for the first antibody or antigen.

The present invention can further be defined in any of the followingnumbered paragraphs:

-   -   1. A method of treating a subject with ischemia, or a subject        after an ischemic injury, the method comprising administering to        a subject a composition comprising at least one peptide selected        from the group consisting of:        Ac-KKKRKV-(βA)-TVTIFVAGVLTASLTIWKKMG-COOH;        AC-PKKKRKV-(βA)-TLKMSVQPTVSLGGFEITPPVVLRLK-COOH (peptide #2) and        AC-PKKKRKV-(βA)-ESFKKQEKTPKTPKGPSSVEDIKAK-COOH (peptide #3), or        functional variants thereof.    -   2. A method for inhibiting the formation of a nucleosphosmin        (NPM)-Bax complex, the method comprising contacting a cell with        at least one peptide selected from the group consisting of:        Ac-KKKRKV-(βA)-TVTIFVAGVFTASFTIWKKMG-COOH;        AC-PKKKRKV-(βA)-TFKMSVQPTVSFGGFEITPPVVFRFK-COOH (peptide #2) and        AC-PKKKRKV-(βA)-ESFKKQEKTPKTPKGPSSVEDIKAK-COOH (peptide #3), or        functional variants thereof.    -   3. A method for inhibiting stress induced cell death, the method        comprising the method comprising contacting a cell with at least        one peptide selected from the group consisting of:        Ac-KKKRKV-(βA)-TVTIFVAGVFTASFTIWKKMG-COOH;        AC-PKKKRKV-(βA)-TFKMSVQPTVSFGGFEITPPVVFRFK-COOH (peptide #2) and        AC-PKKKRKV-(βA)-ESFKKQEKTPKTPKGPSSVEDIKAK-COOH (peptide #3), or        functional variants thereof.    -   4. A method to inhibit nucleosphosmin (NPM) forming a complex        with Bax, the method comprising contacting a cell with an agent        which inhibits the phosphorylation of any of T86, S88, T95, T232        or S240, thereby inhibiting the formation of a NPM-Bax complex.    -   5. A method of treating a subject with ischemia, or having had        an ischemic injury, the method comprising administering to a        subject a composition comprising an agent which inhibits the        phosphorylation of any of T86, S88, T95, T232 or S240, thereby        inhibiting the formation of a NPM-Bax complex.    -   6. A method for inhibiting stress induced cell death, the method        comprising contacting a cell with an agent which inhibits the        phosphorylation of any of T86, S88, T95, T232 or S240, thereby        inhibiting the formation of a NPM-Bax complex.    -   7. The method of any of paragraphs 4-6, wherein the agent is at        least one peptide selected from the group consisting of:        Ac-KKKRKV-(βA)-TVTIFVAGVLTASLTIWKKMG-COOH;        AC-PKKKRKV-(βA)-TLKMSVQPTVSLGGFEITPPVVLRLK-COOH (peptide #2) and        AC-PKKKRKV-(βA)-ESFKKQEKTPKTPKGPSSVEDIKAK-COOH (peptide #3), or        functional variants thereof.    -   8. The method of any of the above, wherein the peptide is fused        to a renal targeting nuclear localization sequence (NSL).    -   9. The method of any of the above, wherein the peptide is        administered within 1-2 days of an ischemic event.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydisclosed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredisclosed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments disclosed herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure. The present invention is further illustrated by thefollowing Examples. These Examples are provided to aid in theunderstanding of the invention and are not construed as a limitationthereof.

EXAMPLES

The examples presented herein relate to the methods, kits, andcompositions for diagnosing a subject with ischemic AKI and methods oftreatment of ischemic AKI using NPM inhibitor peptides a describedherein. Throughout this application, various publications arereferenced. The disclosures of all of the publications and thosereferences cited within those publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Materials and Methods

Animals

All animals were maintained under the guidance and policies of theBoston University Animal Core facility using National Institutes ofHealth (NIH) and Institutional Animal Care and Use Committee guidelines.Male and female C57BL/6J mice were purchased from Jackson Laboratory(Bar Harbor, Me.), and they were housed and bred at the BostonUniversity Animal Core facility under license number AN-15024.Four-week-old mice with mature kidneys afford more consistent resultswith regard to AKI severity and have superior survival after anesthesia.Animas were handled in a manner consistent with the NIH guide for theircare and use under Institutional Animal Care and Use Committee approval.

Cell Culture

Primary culture of human donor kidney and murine kidney proximal tubularcells was performed as previously described. 13,24 Briefly, renalcortices were harvested. Individual cortical samples were minced anddigested by collagenase IV (1 mg/ml); then, they were plated ontoculture dishes in a renal epithelial cell-compatible medium (ATCCPCS-400-030). Cultures of cells that exhibited proximal tubulecharacteristics were maintained for 5-7 days in 5% C02 at 37° C.13,24,25 HEK293NT lentiviral packaging cells (System Biosciences,Mountain View, Calif.) were maintained in DMEM with high glucose medium(Life Technologies BRL, Carlsbad, Calif.) containing 10% bovine serumand 10% penicillin-streptomycin at 37° C. in a 5% CO2incubator. IschemicStress

In Vitro

Exposure to metabolic inhibitors is an established model of ischemicstress that reduces ATP content to, 10% of baseline values untilrecovery is initiated.24,26 To achieve ATP depletion, cells were washedthree times in glucose-free DMEM (Invitrogen, Carlsbad, Calif.) followedby incubation in glucose-free DMEM containing sodium cyanide (5 mM) and2-deoxy-glucose (5 mM) for the indicated times.26

Recovery was initiated by replacing the stress medium described abovewith complete primary cell culture medium containing glucose andsubstrates that support the rapid restoration of cell ATP.27

In Vivo

Mice were subjected to bilateral renal pedicle clamping after beinganesthetized with 2,2,2-tribromoethanol. A midline laparotomy wasperformed, and nontraumatic vascular clamps were placed on both renalpedicles for 25-30 minutes, an insult that produces severe AKI. 13,28,29Slight variation in the clamp time was required to achieve similardegrees of renal dysfunction in the relatively large number of animalsstudied. The clamp was then removed, and reperfusion was confirmed byvisual inspection of the kidneys. Sham ischemia was performed byencircling each renal pedicle with a nonocclusive ligature. The firstpostischemic urine was collected by gentle compression of the inferiorabdominal wall.

Hypoxic Stress

To simulate insults encountered during clinical AKI, PTECs weremaintained under hypoxic conditions for 70 minutes as previouslydescribed, and this is sufficient to induce cell death. 15 Briefly,cells were washed with PBS, transferred to a sealed anaerobic chamberfilled with 95% nitrogen and 5% C02 and incubated in Krebs-Ringerbicarbonate buffer (catalog no. k4002; Sigma-Aldrich). This buffer waspre-equilibrated with 95% nitrogen and 5% C02.

Human Kidneys

Samples of renal cortex were harvested for primary cell culture, andassessment of cytosolic NPM content from paired, non-transplantablehuman kidneys in transportable chambers perfused with chilled Universityof Wisconsin machine preservation solution at 4° C. was performed.30 Inboth kidney pairs, one kidney was normally perfused, whereas the otherexperienced perfusion failure and was, therefore, subjected to aprolonged period of warm ischemia.

Mapping of NPM Phosphorylation Sites by Mass Spectrometry

NPM was purified from human and murine primary renal cells and freshkidney tissue homogenates as well as control and postischemic urine byimmunoprecipitation using an established method. 13 Enzyme digestionwith glutamic acid and/or trypsin was performed to obtain fragmentsappropriate for subsequent analysis. Tandem mass spectrometry spectraobtained by fragmenting a peptide by collision-induced dissociation wereacquired using a capillary liquid chromatography/tandem massspectrometry system that consisted of a Surveyor HPLC pump, a SurveyorMicro AS autosampler, and an LTQ linear ion trap mass spectrometer.Detection and mapping of phosphorylation sites were achieved by databasesearching of tandem mass spectra of proteolytic peptides with specifiedphosphate modification (P=+80 D) on serine, threonine, and tyrosineresidues against the current mouse and human protein databases. Adetailed liquid chromatography/tandem mass spectrometry approach forphosphoproteomics has been previously described.31-33

Mass spectrometry was performed in primary human and renal cells atbaseline and after simulated ischemic stress, in fresh human and murinekidney tissue homogenates before and after frank ischemia, and in humanand murine urine from patients without AKI and patients with AKI beforeand after experimental AKI, respectively. At least three replicates ofeach purified NPM sample harvested from renal cells, kidney tissue, andfresh urine were subjected to mass spectrometry.

NPM Phosphomutant Construction and Infection

Human NPM cDNA (catalog no. 34553; Addgene, Cambridge, Mass.) and pCDHlentivector systems (System Biosciences, Mountain View, Calif.) wereused to generate NPM wild-type, “NPM stress mimic”(T86E-S88E-T95E-T234A-S242A), and “NPM normal mimic”(T86A-S88A-T95A-T234E-S242E) lentiviral expression plasmids by PCR-basedmutagenesis with a flag tag fused to the 59 open reading frame. PCRproducts were cloned into pCDH vector BamHI/EcoRI sites. All constructswere confirmed by DNA sequencing before cotransfection with pPACKG1lentivector packaging plasmids into HEK293NT cells to generatelentipseudoviral particles. The resultant pseudoviruses were harvestedand purified, and titers were determined according to the manufacturer'sinstructions. Cells were exposed to virus (MOT 5-8) diluted in Opti-MEM(Invitrogen). After 6 hours incubation with virus-infected medium, acomplete culture medium was substituted for an additional 16-hourincubation period.

NPM Knockdown

Clustered regularly interspaced short palindromic repeats (CRISPR)interference was used to transiently suppress NPM. After 60 hours underroutine cell culture conditions, primary cells were transfected with twosgRNA targeting adjacent to the NPM transcription start site(synthesized by Synthgo, Menlo Park, Calif.) using TransIT-2020Transfection Reagent using the manufacturer's protocol. Six hours later,five MOI of dcas9-KRAB lentivirus (catalog no. k204; abm.com, Richmond,BC, Canada) were added. The medium was then changed, and culturecontinued for 48 hours. The magnitude of NPM suppression was assessed byimmunoblot analysis.

Dot Blot Analyses

Because relatively low NPM concentrations in urine are insufficient forroutine immunoblot due to volume limitations of sample loading, a dotblot assay standard method and protocol were used(http://www.abcam.com/ps/pdf/protocols/dot%20blot%20protocol.pdf). Inthe dot blot assay, NPM can be easily detected, because a much largerurine volume can be loaded. Importantly, identical anti-NPM antibodieswere used in both the immunoblot and dot blot assays.

Renal-Targeted NPM Peptides

For in vitro testing in primary human PTECs, peptides (200 mM) weretested that replicate the Bax domain responsible for binding NPM,34 ortwo peptides designed to interfere with NPM function were compared witha random nonspecific sequence of the same length (control). Peptidesintended to interfere with NPM function overlap key phosphorylationsites located at the NPM amino terminus (peptide 2) or carboxy terminus(peptide 3) detected by mass spectrometry. To improve cell uptake, allpeptides contained a cell-penetrating sequence13. For in vivoexperiments, a renal-targeting sequence (NLS) was added to enhance renalpeptide uptake.35 Peptides were commercially synthesized (Biomatik,Wilmington, Del.) J Am Soc Nephrol 30: ccc-ccc, 2018 Nucleophosmin inAKI 3www.jasn.org BASIC RESEARCHas follows: NPM-Bax blocking peptide:Ac-KKKRKV-(bA)-TVTIFVAGVLTASLTIWKKMG-COOH (SEQ ID NO: 8); peptide 2:AC-PKKKRKV-(bA)-TLKMSVQPTVSLGGFEITPPVVLRLK-COOH (SEQ ID NO: 9); andpeptide 3: AC-PKKKRKV-(bA)-ESFKK-QEKTPKTPKGPSSVED-IKAK-COOH (SEQ ID NO:10). A single dose (100 mg/g body wt of the NPM-Bax blocking or controlpeptide) was administered by tail vein injection immediately afterrelease of the pedicle clamps(0 timepoint) as well as 1,2,3,4, 5, and 6hours later in each experimental group. A total of 42 control and 56experimental animals were studied.

Cell Viability or Kidney Function

Transient exposure to metabolic inhibitors replicates ischemia in vivoby activating Bax and causing cell death.13,29,36-38 Cell viability wasassayed using a modified colorimetric technique (MTTassay).37 The numberof surviving cells is expressed as a percentage of viable control cellsdetected at baseline. Serum BUN and creatinine levels were measured intail vein blood samples for 7 days after ischemia using QuantiChrom BUNor creatinine assay kits (BioAssay Systems, Hayward, Calif.) accordingto the manufacturer's instructions.

Protein Isolation and Immunoprecipitation

Cell, tissue, and urinary proteins were harvested using PD buffercontaining 40 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Nonidet P-40, 6 mMEGTA, and a protease inhibitor cocktail (Set I; Calbiochem, San Diego,Calif.). NPM was immunoprecipitated overnight with NPM antibodies(catalog no. B0556; Sigma-Aldrich) in the presence of proteinA/G-agarose beads at 4° C. NPM accumulation was measured in cytosolicsamples extracted with low-dose digitonin (8 mg/ml).13 Cytosolicproteins were extracted from renal cortical tissue samples using anextraction kit (Biochain Institute, Newark, Calif.). Briefly, 50-100 mgof kidney cortex tissue was minced on ice and spin washed with 1 mlice-cold wash buffer before adding 100-200 ml of buffer C. The mixturewas rotated at 4° C. for 20 minutes and then centrifuged at 18,0003 g at4° C. for 20 minutes before being harvested. Protein levels weremeasured with the bicinchoninic acid assay (Thermo Scientific, Rockford,Ill.).

Immunohistochemistry

NPM was visualized as previously reported, 13 and the protocol wasperformed according to the manufacturer's protocol (catalog no. B0556).

Dot Blot Assay

NPM was detected in murine urine using a dot blot assay as previouslyreported39 using an antibody directed against total NPM.

Statistical Analyses

Data are expressed as means with SEM. Differences between groups weredetermined by a two-tailed t test using Excel (Microsoft, Redmond,Wash.). Comparisons involving more than two groups were determined by atwo-way ANOVA followed by a Holm-Sidak post hoc test for nonparametricdata. P, 0.05 was considered significant.

Example 1 Stress Causes NPM Translocation in Both Cells and Tissues

NPM translocation from the nucleus to the cytosol promotes celldeath.40,41 To determine the extent to which ischemic stress causes NPMtranslocation, an early step in the NPM-mediated death pathway,42 PTECswere subjected to transient ATP depletion, an established model ofmetabolic stress that resembles ischemia.24,26 In resting primary humanPTECs, 0.95% of total cell NPM localized to the nuclear (i.e.,noncytosolic) fraction. In contrast, about 15% of total cell NPMaccumulated in the cytosol after stress. Compared with normal cells,only about 80% of NPM remained intracellular (15% cytosolic plus 65%nuclear) (FIG. 1A), demonstrating that extracellular NPM leakage or NPMdegradation occurred. Stress caused marked cytosolic NPM translocationin ATP depleted primary murine PTECs (FIG. 1B) or renal homogenatesharvested from grossly ischemic, nontransplantable human kidneys ex vivocompared with well perfused kidneys harvested from the same donor (FIG.1C).

Cytosolic NPM translocation was also detected in murine and humanprimary PTECs after transient hypoxia (FIG. 1D). Densitometric analysisconfirmed the magnitude and reproducibility of NPM translocation. Bothde-energization and hypoxia contribute to renal epithelial cell injuryand organ failure during human AKI.3

Stress Induces Site-Specific NPM Phosphorylation Changes

To identify the post-translational events that regulate NPM toxicity,the effect of metabolic stress on differential NPM phosphorylation wasexamined by mass spectrometry in purified NPM harvested from human andmurine kidney cortex and human and murine primary PTECs after ischemicstress as well as after murine and human AKI. Representative tracingsare shown (FIG. 2A-2C). Digestion of NPM from cells and tissue withtrypsin and glutamic acid afforded positive identification of 269 of 292(92%) potential NPM residues and yielded 100% all known serine,tyrosine, and threonine residues capable of undergoing ischemia-inducedphosphorylation or dephosphorylation. Despite a two-amino aciddifference in overall length, the amino acid sequences are >94% similarin murine and human NPM, and the phosphorylation consensus sequences areidentical. Compared with control renal tissue and cells, five distinctphosphorylation changes at T86, S88, T95, T234, and S242 were observedafter ischemic stress (FIG. 2D). Remarkably, only a single event(dephosphorylation at T86 in murine kidney tissue) differed fromsite-specific phosphorylation changes detected in other cell or tissuesamples subjected to ischemic stress. The pattern of urinary NPMphosphorylation identically matched phosphorylation at four of fiveserine/threonine residues detected in ischemic cells and renal tissue(FIG. 2D). Only the peptide fragment containing S240 was not detected inNPM purified from postischemic murine urine. In a patient with KidneyDisease Improving Global Outcomes criteria for stage 3 AKI, 43differential phosphorylation at residues T86, S88, and S242 was detected(FIG. 2D). The pattern of NPM phosphorylation in human urine during AKIresembled NPM phosphorylation changes in ischemic cells, tissue, andmurine urine after experimental AKI. Total NPM was readily detected inmurine urine within 6 hours after renal ischemia and disappeared within48 hours after injury (FIG. 3)

NPM Phosphomimic Proteins Mediate NPM Toxicity

To determine the biologic role of differential NPM phosphorylation atthese five serine/threonine sites and the contribution to cytotoxicity,two flag-tagged NPM phosphomutant proteins as well as wild-type NPM weregenerated and introduced into human proximal tubule cells. These twomimic proteins replicated the phosphorylation state of NPM in either itsnormal or stressed configuration at the five differentiallyphosphorylated sites identified by mass spectrometry (FIG. 4). Specificbioassays were then used to assess the effect of these fivepost-translational phosphorylation changes on toxic NPM behaviors,including translocation, deoligomerization, NPM-Bax complex formation,mitochondrial NPM and Bax accumulation, and cell death. Both thewild-type and normal NPM phosphomimic proteins accumulated in anucleolar pattern within resting cells (FIG. 5A, left and centerpanels). In contrast (and in the absence of ischemic stress), only theNPM stress mimic protein localized to the cytosol (FIG. 5A, rightpanel). In resting cells, wild-type and normal mimic NPM formed largeoligomers. In contrast, the stress mimic NPM protein accumulated assmall monomers known to interact with conformationally active Bax (FIG.5B). After ATP depletion sufficient to activate Bax, NPM-Bax complexformation was substantially greater in cells that contained stress mimicNPM (FIG. 5C). Similarly, the accumulation of both stress mimic NPM andactive Bax accumulation in isolated mitochondria after ischemic stressexceeded that observed with wild-type or normal mimic NPM (FIG. 5D).Compared with control or cells that expressed either empty vector (anadded transfection control) or wild-type NPM, postischemia survival wassignificantly lower in cells containing stress mimic NPM (FIG. 5E)(P,0.05). Unexpectedly, normal mimic NPM expression significantlyreduced cell death (P,0.05). Compared with wild-type NPM, the expressionof normal mimic NPM also decreased NPM-Bax complex formation (FIG. 5C)and the accumulation of NPM and Bax in isolated mitochondria afterischemic stress (FIG. 5D).

Example 2

NPM is a Relevant Target for Ameliorating Renal Cell Injury

To assess the causal role of NPM in renal cell death, primary humanPTECs were subjected to ischemic stress before and after NPM knockoutusing an inducible CRISPR system. CRISPR reduced NPM expression by80%-90% (FIG. 6, inset) and significantly increased cell survival afterischemia (FIG. 6).

Cell and Animal Survival after Ischemic Stress is Improved by PeptidesDesigned to Interfere with NPM Function

Identification of the key events that regulate NPM cytotoxicity raisedthe possibility that peptides designed to interfere with stress-inducedNPM phosphorylation or NPM-Bax complex formation might have therapeuticpotential. To test this hypothesis, peptides that replicate thephosphorylation consensus sequences altered by ischemic stress (FIG. 2D)at the NPM amino (amino acids 78-103) or carboxy termini (amino-acids226-246) or likely to interfere with NPM-Bax complex formation34 weregenerated and tested. After introduction into human PTECs, all threepeptides significantly improved post-ischemic cell survival (FIG. 7). Toassess its efficacy in treating severe ischemic AKI, a single peptidedose was intravenously administered to mice 0-3 hours after releasingbilateral renal pedicle clamps after 28 minutes of ischemia, an insultsufficient to induce lethal organ failure. All control mice thatreceived a random peptide (six of six) died within 72 hours afterpedicle clamp release (FIG. 8A). Before death, these animals had BUNconcentrations that averaged 120 mg/dl, a marked degree of azotemia. Incontrast, animals receiving a single dose of a renal targeted peptidedesigned to interfere with Bax-NPM complex formation exhibitedsignificantly lower BUN levels and less severe organ failure on days 1-7post-ischemia. Peptide treatment also reduced serum creatinine by nearly50% on day 1 post-ischemia, a time point associated with marked BUNaccumulation (FIG. 8B). Remarkably, seven of eight peptide-treatedanimals (88%) survived severe ischemic AKI. Peptide administration at 0,1, or 2 hours post-ischemia also provided significant protection againstlethal renal ischemia (P,0.05 versus control), but no protection wasobserved if the same peptide dose was administered 4, 5, or 6 hoursafter transient renal ischemia (P.0.05) (Table 4). In peptide-treatedanimals, recovery of renal function was complete, and it was sustainedfor at least 4 weeks. Blocking peptide treatment markedly reducedBax-NPM interaction in renal epithelial cells in vitro (FIG. 9A) as wellas postischemic renal homogenates (FIG. 9B).

TABLE 4 Renoprotective effect of peptide therapy for AKI. Summary ofanimal survival data after transient renal ischemia; experimentalconditions were identical to those described in FIG. 8. A single dose ofblocking peptide or a control peptide was administered at 0, 1, 2, 3, 4,5, and 6 h after AKI induction. Animal deaths were presumed due to AKI,and they were recorded for 7-d post-pedicle clamp release; no deathswere observed after 3 d. Significant protection by the blocking peptideon animal survival was observed for doses administered at 0, 1, 2, or 3h post-AKI (P, 0.05); the blocking peptide was not protective ifadministered at 4, 5, or 6 h post-AKI (P.0.05). Peptide Treated, n = 8Animals at Control, n = 6 Animals at Each Each Time Point (DosePost-AKI, h) Time Point (Dose Post-AKI, h) Days (Post-AKI) 0° 1° 2° 3°4° 5° 6° 0° 1° 2° 3° 4° 5° 6° 1  1  1 1  1  1 2  1 1 1 2  1 2  1  1  1 4 6  4 4 3  4 2 3 4  4 3 3  2 3 3 2 Total deaths  1  2  1  1 8  7  7 6 6 5 6 6 6  5 Survival , % 88 75 88 88 0 13 13 0 0 17 0 0 0 17

Example 3

Herein, the inventors have discovered that post-translationalmodifications convert NPM from a highly conserved protein that isessential to cell survival to a cytotoxin and demonstrate that thesepost-translational phosphorylation changes link early AKI diagnosticswith effective therapeutics. Mass spectrometry of purified NPM revealsfive distinct serine/threonine sites that regulate NPM toxicity duringischemic stress in both primary renal cells and intact tissues of twogenetically divergent mammals, showing conservation of this mediator inthe stress-induced cell death pathway. Expression of an NPM phosphomimicprotein that replicates the differential phosphorylation changesdetected in primary renal cells, tissue, and urine after an ischemicinsult reproduces NPM translocation and deoligomerization, key behaviorsof the wild-type NPM observed during ischemic stress. Interestingly, twoof five phosphosites identified in herein (S88 and T95) also regulateNPM deoligomerization and cytosolic translocation in human cancer cells,respectively,40,41 and they determine responsivity to therapy inpatients with acute myelogenous leukemia by enhancing their sensitivityto chemo- and radiation-induced apoptosis.44,45

Semi-quantitative analysis of peptide abundance by mass spectrometryrevealed that >80% of cytosolic NPM was differentially phosphorylated inthe toxic pattern after stress. Thus, the inventors have demonstratedthat differential phosphorylation positively correlates withtranslocation to the cytosol. In addition to S88 and T95, the inventorshave also discovered three other stress-induced differentialphosphorylation changes that regulate NPM toxicity in ischemic renalcells (outlined in FIG. 10). The proposed intracellular events are onthe basis of the discovery that the NPM phosphomimic protein (with allfive stress-induced phosphorylation changes) undergoes (1) cytosolic NPMtranslocation (FIG. 1 and FIG. 5A), (2) deoligomerization of large NPMpentamers to monomers capable of traversing the nuclear pore46 (FIG.5B), (3) complex formation with conformationally activated Bax (FIG.5C), and (4) coaccumulation with Bax in isolated mitochondria (FIG. 5D)and cell death (FIG. 5E).

Alterations in NPM phosphorylation alone, even in the absence of cellstress, are sufficient to cause cytosolic NPM accumulation anddeoligomerization. In contrast, NPM-Bax complex formation andmitochondrial NPM-Bax accumulation require both site-specific NPMphosphorylation changes and stress-induced conformational Baxactivation. The inventors previously reported that mitochondrial Baxaccumulation is NPM dependent 13 and that the NPM-Bax complex forms onlyafter Bax undergoes conformational activation, 13,34,47 exposing theamino-terminal 6A7 epitope.48,49 Although increasing cytosolic NPMsignificantly promotes Bax-mediated cell death, increasing cytosolic NPMper se is nontoxic. 13 These inventors demonstrate herein that both NPMand Bax are both required to cause stress-induced cell death, and theyare analogous to Knudson “two-hit hypothesis” of cancer celltransformation.50 In a biologic twist of fate, Bax has also beenimplicated in releasing nuclear NPM into the cytosol,51 further linkingtheir toxic behaviors. Effective reagents in our experimental models arelikely to be therapeutic for Baxmediated renal injury in humans, becausevirtually identical post-translational NPM modifications at identicalconsensus sequences were detected in ischemic murine and human cells andtissue and were partially replicated in urine during clinical andexperimental AKI.

Several laboratories have reported that Bax-regulating kinases,including protein kinase B (Akt13,52), glycogen synthase kinase 3b13,53,and Jun N-terminal kinase,54 activate Bax. In renal cells,conformational Bax activation is primarily due to the loss ofAkt-mediated Bax serine 184 phosphorylation during ischemic stress. 13Surprisingly, the normal NPM phosphomimic protein significantly improvedcell survival after stress. This is likely due to the fact the normalphosphomimic protein inhibited both stress-induced NPM-Bax complexformation (FIG. 5C) and NPM-Bax accumulation in isolated mitochondria(FIG. 5D) compared with either the wild-type or stress mimic NPM.Accordingly, the inventors have demonstrated herein that the normal NPMphosphomimic mutant protein acts as a competitive inhibitor ofendogenous NPM and may itself be a therapeutic agent.

Herein, the inventors demonstrate that differential NPM phosphorylationis a potential urinary marker for renal epithelial cell injury as wellas an effective target for peptide-based therapeutics directed againstNPM. Within hours of renal ischemia (and at the time of first urinecollection), both total and phosphorylated urinary NPM are readilydetectable. This is more likely due to NPM leakage across the cellmembrane than NPM degradation during the brief period of ischemia. 13Herein, the inventors demonstrate using mass spectrometry that urinaryNPM exhibits the same pattern of phosphorylation and dephosphorylationas NPM harvested from renal cells and fresh issue. The discovery thatboth total and phosphorylated NPM are detectable within hours afterrenal ischemia, well before a rise in serum BUN/creatinine is evident,further validates NPM phosphorylation states as early AKI markers thatcan be used to initiate effective AKI treatment. Despite marked organdysfunction, total NPM is no longer detected in urine 48 hours after theinsult, demonstrating that it reflects acute renal epithelial cellinjury. Although total urinary NPM could result from detached,sublethally injured renal cells, the phosphorylated NPM is specific forlethal cell injury. This is strongly demonstrated herein which showsthat wild-type NPM overexpression in renal cells is nontoxic, 13 whereasstress phosphomimic NPM exacerbates ischemic cell death. The magnitudeof differential NPM phosphorylation during ischemic stress is uncertainand will likely require quantitative mass spectrometry to be adequatelyaddressed. In biology, it is unusual for protein to exist in a singleform, because phosphatase and kinase activity act as opposing forces toregulate protein function.55 On the basis of compelling data gleanedfrom the phosphoproteomic study and peptide treatment, the inventorshave discovered that NPM is a marker of acute renal cell injury that canbe physiologically linked to effective AKI treatment.

NPM has been proposed by others to be a Bax chaperone,34,47 and it isstrongly implicated in regulating Bax-mediated ischemic renal cellinjury. 13 However, herein, the inventors demonstrate that a NPMblocking peptide (e.g., also called a NPM inhibitory peptide) designedto interfere with NPM-Bax interaction decreases NPM-Bax complexformation in vitro and in vivo by about 50% (FIG. 9), improves renalcell survival (FIG. 7), and substantially improves kidney function(i.e., reduced both BUN and creatinine) by 50%. While a single effectiveinhibitor is likely to afford only partial protection, a cocktail ofsite-specific peptides directed at multiple steps in the NPM-Bax celldeath pathway could improve renoprotection. The discovery that NPMphosphorylation has interdependent regulatory effects on oligomerassembly and protein partner binding20 (e.g., with Bax) supports the useof multiple reagents. Infact, such strategies are useful if NPM- andBax-independent forms of cell death contribute to organ failure afterischemia.56 However, substantial evidence suggests that Bax contributesto the forms of regulated cell death most often detected after renalischemia, including apoptosis, necrosis, 57-59 and more recently,necroptosis, a biochemically distinct form of cell death.57,60

Inhibitory or blocking Peptides are ideal for treating renal diseasesthat primarily target the PTEC. Peptides have few untoward side effectspartly due to their extremely short serum t1/2.61 Furthermore, peptidesthat contain the NLS sequence selectively accumulate in the kidney. Infact, 94% of an intravenous radiolabeled peptide attached to the NLSsequence accumulates in the murine kidney.35 It is highly likely themegalin receptor, located on the brush border of the PTEC, mediatesrenal peptide uptake and markedly prolongs its t1/2.62 Herein,NLS-containing NPM inhibitor peptides of SEQ ID NO: 1-3 were uniquelydesigned to interfere with separate NPM functions, and were demonstratedto significantly protect cells against ischemic stress (FIG. 7) aseffectively as CRISPR-mediated NPM knockdown (FIG. 6). The 20%-30%improvement in cell survival is likely to significantly promote organrecovery by more rapidly replacing cells lost during ischemia, a keydeterminant of organ recovery after AKI.28,63 In contrast to universallylethal AKI in control, 75%-88% of animals survived after a single doseadministration of a NPM inhibitor peptide, administered 0-3 hours afterreleasing the renal pedicle clamps, demonstrating significant efficacyof the NPM inhibitor peptides in treating ischemic AKI in vivo, which isa disease the presently lacks an effective treatment. Although NPMinhibitor peptide administration did not completely protect against AKI,treatment was sufficient to promote animal survival, equivalent toavoiding RRT in human AKI. The 3-hour therapeutic window for peptidetreatment is similar to that reported for thrombolytic treatment inacute myocardial infarction64 and ischemic stroke,65 insults in whichBax-mediated apoptosis and necrosis have been reported. 58,66-68Although relatively narrow, this treatment window exceeds that reportedfor other forms of programmed cell death in renal ischemiareperfusioninjury, including necroptosis, that can only be given before the insult.69

The remarkable efficacy of interfering with a single-cell death pathwayis likely due to the facts that (1) Bax-mediated apoptosis and necrosisrepresent a continuum of cell death rather than distinct pathways13,70,71,72; (2) cytosolic NPM translocation, deoligomerization, andNPM-Bax complex formation occur before cells are irreversibly committedto die13,34 (unlike blocking downstream caspases); and (3) renal celldeath is partly responsible for the subsequent oxidant injury andinflammation that accompany organ failure.72 As a result, early AKIdetection linked with early treatment is especially attractive.

In response to stress, approximately 75%-80% of cell Bax colocalizeswith mitochondria without a known mitochondrial localizing sequence74 oran identified outer membrane receptor, 73 indicates that Bax requires acytosolic chaperone to target mitochondria. Herein, the inventors havedemonstrated that virtually identical phosphorylation changes atspecific amino acid residues convert NPM from an essential cytosolicprotein to a toxic Bax chaperone in both mice and humans and thephosphorylation state of the NPM polypeptide is an early marker ofischemic renal cell injury and is also a target for therapeuticintervention. The presence of both NPM and Bax in all mammalian cellsindicates that this regulatory cascade also participates in tissueinjury in other ischemia-susceptible organs.

Example 4

Nucleophosmin (NPM) Phosphorylation States as a Diagnostic for AKI andNPM Peptides as Therapeutic Treatment of Subjects with AKI

Nucleophosmin (NPM) translocation from the nucleolar region of thenucleus into the cytosol or extracellular compartment (blood or urine)heralds cell death mediated by Bax, a toxic member of the BCL2 proteinfamily that regulates apoptosis and regulated necrosis. Changes in NPMphosphorylation status at 5 specific sites alters NPM function andregulates early steps in the death pathway in mammalian cells subjectedto stress. Peptide reagents directed against NPM reduce its toxicity,preserve organ (renal) function, and significantly improve animalsurvival if administered either before or within 4 hours after anischemic insult. Similarity between human and murine NPM sequencesallows for rapid translation to human disease. Mass spec revealsidentical NPM phospho-changes in human and murine cells subjected to ATPdepletion as well as in post-ischemic murine and human kidneys. NPMtranslocates into the cytosol and urinary lumen of proximal tubule cellsin biopsy specimens obtained from humans with acute tubular injury andAKI and transiently appears in the urine of mice subjected to ischemicAKI before changes in renal function occurs as measured by traditionalBUN/Cr assays. New bioassays will be linked to treatment with novelanti-NPM peptide(s). The inventors use immunoassays, e.g., ELISA tomeasure both NPM and phospho-NPM in fluids and use site specificphopsho-antibodies to detect NPM in various stages of activation thatresult in renal and non-renal cell death.

The inventors herein have assessed individual labeled NPMphospho-proteins by Mass Spec to assess their contribution to NPMfunction and cell death in the presence or absence of endogenous NPM.Phospho-specific antibodies allow tire phosphorylation status of NPM tobe detected in tissues to determine the severity of tissue injury andpredict prognosis. In particular, the detection of total nucleophosminand nucleophosmin phosphorylation status in the blood, urine and humantissues before, during and after ischemic insults can acts as adiagnostic, as well as determines the severity of injury and determinesprognosis (likelihood of organ recovery), and also guide therapeuticinterventions that minimize cell death and gauge treatment efficacy,e.g., determine therapeutic efficacy of cancer treatments in real timebefore current clinical tests reveal tissue injury.

The inventors have discovered that the different phosphorylation statesof the NPM polypeptide is useful for (1) assessing and testing for acutestress (e.g, ischemia), which is presently unavailable and occurs beforecurrent measures of tissue (e.g., kidney), (2) linking NPM diagnosticsto NPM inhibitor peptide therapeutics that prevent or treat ischemicinjury and kidney injury, and/or NPM mediated cell death, (3) predictthe severity and prognosis of acute tissue injury, and (4) detection ofNPM leakage into blood and/or mine represents a novel measure of acutecell death (a primary and desirable endpoint) in cancer patients. In anexemplary use, detection of the changes in NPM phosphorylation can beused to predict cell death in non-kidney tissues and blood, as well asAMI, prognosis.

Another exemplary use of the diagnostic assays described herein isdetection of the phosphorylation state of the NPM polypeptide as ameasure of cancer therapy efficacy. For example, monitoring or detectingchanges in the phosphorylation state of the NPM polypeptide in blood andurine biological samples can be used as a measure cell death duringchemo- or radiation therapy. For example, urinary NPM may be a marker ofgeneralized cell death during chemotherapy and evidence of effectivetreatment, as well as the pattern of NPM-phosphorylation in urine usedto reflect cell killing by apoptosis (near 30 kDa MW filtration cutoff)during cancer treatment.

NPM Peptide Assays & Therapeutics

Small NPM inhibitor peptides (20-30AA size) can be fused to a renalspecific cell penetrating peptide to markedly increase renal delivery(90%). The NPM inhibitor peptides are directed at blocking Bax-NPMinteraction and/or NPM toxicity related to phosphorylation changes at 5sites, and a single peptide dose was demonstrated to be effective atpreventing ischemic-induced cell death, and there was no detectabletoxicity observed in mice in vivo.

Herein, the inventors have also demonstrated that in a preliminarystudy, peptide(s) that mimic NPM-phosphorylation sites improve survivalin renal epithelial cells subjected to ATP depletion, and that suchpeptides may be useful for preventing stress-induced cell death in thekidney and other organs.

The inventors also demonstrate use of functional assays that to measurephosphorylation at each of 5 NPM sites alterations, which can be used toassess its toxicity with regard to any one of: NPM nuclear release,de-oligomerization, NPM-Bax interaction, NPM-Bax complex translocationto mitochondria, and mitochondrial injury)

The therapeutic NPM inhibitory peptides directed against NPM are alsoassessed by their functional role and its relative toxicity determinedby distinct NPM assays of site-specific NPM phospho-changes

Herein, the inventors demonstrate NPM toxicity is amenable to NPMinhibiting peptides that act that competitive inhibitors and interferewith NPM-Bax interaction (required for cell death) and/orNPM-phosphorylation that mediates toxic NPM behaviors (release from thenucleolar region, oligomerization, cytosolic translocation, interactionwith Bax, and migration of the NPM-Bax complex to mitochondria), theprimary target of cell death. Since Bax mediates stress-induced celldeath in the heart, brain, kidney, liver and bowel it is likely thatpeptide therapeutics targeted to these organs will ameliorate acuteischemic tissue injury after a myocardial infarction, stroke or after anischemic insult to the kidney, liver, or bowel. Since NPM is highlyconserved in nature, it is likely that multiple stressors and tissuesutilize a NPM-Bax mediated cell death pathway. Therefore, maneuvers thatinterfere with NPM-Bax function are highly likely to be effectivetherapeutics.

As such, the inventors have discovered that NPM phosphorylation statesis useful in diagnostic testing for acute stress (e.g, ischemia) beforecurrent measures of tissue (e.g., kidney) injury, and that NPMdiagnostics can be linked to treatment using the NPM inhibitorsdescribed herein, e.g., the NPM inhibitor peptides that prevent or treatNPM-mediated cell death and prevent or treat tissue injury. Moreover,the inventors have demonstrated that measuring NPM phosphorylation andcell localization are useful to predict the severity and prognosis ofacute tissue injury, and that NPM leakage into blood and/or urinerepresents a novel measure of acute cell death (a primary and desirableendpoint) in cancer patients. In particular, NPM phosphorylation andurinary NPM can be used for titration of chemo- and radiation therapies.In some instances, detection of urinary NPM can be used to determine ifthe subject should discontinue the nephrotoxic medications (includingcancer treatments) or limit procedures in which renal ischemiapredictably occurs. In another instance, the leakage of NPM orphospho-NPM into the serum can be used as a biomarker of myocardialinfarction (MI) and/or ischemic stroke and can be used to determine theseverity and/or prognosis of each.

Example 5

As disclosed in Examples 1-2, the NPM inhibitory peptide (including therenal targeted peptide) decreased NPM-Bax complex formation in thepost-ischemic renal cortex in vivo, with improved organ function. Thisdata clearly demonstrates the therapeutic efficacy of antagonizingNPM-Bax interaction during ischemia and invites additional interventionsthat reduce NPM-Bax toxicity to prevent and treat AKI. Accordingly, theinventors demonstrate that NPM post-translational phosphorylation eventsalter NPM function and contribute to renal epithelial cell death inischemic human kidney tissue. Inhibition of the NPM-Bax complex istherefore useful in treating ischemic-induced cell stress and AKI. Inparticular, detection of total NPM protein and the phosphorylated stateof the NPM protein in urine is useful as an AKI biomarker and can beused to guide effective drug dosing of treatment for AKI and/or cancertherapies.

As discussed herein, NPM is an abundant, highly conserved nucleolarchaperone that is essential for normal cell function. In patients withacute myelogenous leukemia (AML), NPM mutations that alter NPMdistribution (i.e., to the nucleus vs. cytosol) determine thesusceptibility to chemo- and radiation therapy and therefore, prognosis.Post-translational phosphorylation sites have been reported to regulateNPM in human cancer cells.

NPM is essential mammalian phospho-protein that normally resides in thenucleolus as a homo-pentamer15,16, a membrane-less region within nuclei.NPM is a molecular chaperone, that shuttles between the nucleus andcytosol to regulate ribosomal biogenesis, protein synthesis, centrosomeduplication, cell cycle progression, and tumor suppression. NPMphosphorylation regulates cell death in both cancer and non-cancercells. Accordingly, by assessing NPM's localization, de-oligomerizationand its chaperone function, as well as its phosphorylation state, NPMcan be used as a biomarker to predict a patient's prognosis by alteringtheir susceptibility to chemotherapy-induced apoptosis.

Bax, a mitochondrial toxic BCL2 protein, mediates both apoptosis andnecrosis and is a major cause of ischemic tissue injury. In the absenceof mitochondrial localizing sequence, 80% of Bax localizes to themitochondrial surface during apoptotic stress, suggesting that Baxrequires a “chaperone service”. Several labs, including our own, haveshown that nucleophosmin (NPM or B23) accumulates in the cytosol ofischemic tissue, binds Bax only its conformationally active form and isa key as a Bax chaperone during ischemic AKI. In renal cells, neitherNPM nor Bax kills alone. Overexpression of conformationally changed(i.e., conformationally active) Bax, characterized by exposure of theamino terminal 6A7 epitope, is non-lethal in the absence of exogenousstress. The inventors demonstrate that after stress, NPM rapidlycomplexes with Bax and localizes to mitochondria and causes cell death.Cell death is markedly enhanced by expressing a cytosol restricted NPMmutant. As such, the inventors demonstrate that cell death requires that“two hits” to form toxic NPM-Bax complexes: conformational Baxactivation and cytosolic NPM chaperone accumulation.

The inventors herein demonstrate that limiting NPM-Bax complex formationameliorates ischemic AKI. Since NPM's structure, functional domains, andregulatory sites are virtually identical in mice and humans, it allowsthe inventors to test novel NPM diagnostics and therapeutics in cells,as well as in intact murine and human kidney tissue. Our centralhypothesis is that NPM-Bax mediated cell death can be effectivelyprevented and treated. This strategy can translate to effective clinicaltrials for human AKI.

Determine the Role of Stress-Induced NPM Phosphorylation Changes inRenal Cell Death and AKI.

In humans, both cell necrosis and apoptosis have been widely implicatedin contributing to ischemic AKI. Bax, a toxic BCL2 member, triggers bothapoptosis and necrosis by permeabilizing the outer mitochondrialmembrane and altering mitochondrial dynamics. However, Bax lacks amitochondrial localizing sequence, suggesting that Bax requires a“chaperone service” to translocate to mitochondria. Herein, theinventors demonstrate that NPM is a primary Bax chaperone that causesrenal cell death that contributes to human ischemic AKI. In human cancercells, the NPM chaperone function is regulated by post-translationalphosphorylation. Herein, the inventors demonstrate that differential NPMphosphorylation during renal ischemia is essential for its chaperonefunction and mediates NPM-Bax toxicity that contributes to human AKI.

Moreover, the inventors demonstrate that NPM and Bax contribute to renalcell death during ischemic stress, and that NPM translocation occurs inboth murine and human PTEC subjected to either ischemic stress (10 mMrotenone) or hypoxia (95% nitrogen 5% CO2 gas for 60 min) (data notshown). NPM translocation is regulated rather than a consequence ofnuclear membrane injury. Overexpression of wild type NPM increasesnuclear NPM content without increasing ATP depletion-induced death. Incontrast, over-expression of cytosol restricted NPM mutant markedlyincreases both mitochondrial NPM and Bax accumulation, AIF andcytochrome c release, caspase 3 activation and death in ATP depletedPTEC in a Bax-dependent manner. Therefore it is cytosolic, rather thannuclear NPM content, that is the rate limiting in stress-induced PTECdeath. In cell lines subjected to ischemic stress, cytosolic NPMtranslocation precedes mitochondrial Bax accumulation and in theischemic kidney, results in persistent NPM-Bax complex formation. Arecent report of multimeric NPM complexes in the nucleolar region ofresting cells indicates that NPM pentamers likely predominate in thenuclear compartment in PTEC, whereas cytosolic NPM monomers accumulateonly after ischemic stress. Fortunately, proximal tubule avidlyconcentrates intravenously administered peptides, a primary target ofinjury in ischemic AKI. Herein, a peptide that disrupts NPM-Baxinteraction by mimicking the NPM binding domain of Bax has been reportedto reduce mitochondrial injury, cell death and protects renal functionand markedly improves animal survival, the murine equivalent ofrequiring renal replacement therapy. As demonstrated herein, thispeptide (peptide #1) markedly decreases NPM-Bax complex formation inPTEC and in murine cortical homogenates harvested after in vivoischemia, respectively. Interestingly, a 50% reduction in NPM-Baxcomplex formation improves renal function after transient ischemia by asimilar degree 1 and motivates the development additional therapeuticsdirected against NPM-Bax.

To determine whether ischemia-induced posttranslational phosphorylationevents regulate NPM-Bax toxicity as described in AML patients, primarymurine and human PTEC, as well as ischemic murine and human kidneytissue (harvested from cadaveric kidneys rejected for transplantation)was subjected to mass spectrometry after protease digestion, a sensitivetechnique identifies site-specific phosphorylation and dephosphorylationevents. Mass spec of NPM purified by IP revealed marked differential NPMphosphorylation in normal vs. ATP deplete primary mouse PTEC; sham andischemic mouse kidney; as well as between paired kidneys harvested from2 donors in which one kidney was normally perfused and other wasischemic. Trypsin plus glutamic acid digestion yielded positiveidentification of 269 of 292 (92%) of NPM residues and remarkably,included 100% all known serine, tyrosine and threonine residues capableof undergoing ischemia-induced phosphorylation or dephosphorylation.

In contrast to the 64 NPM phospho-changes predicted by PhosphoSite.Org,the inventors demonstrate that only 5 NPM serine/threoninephosphorylation events differed between normal and stress conditions.During ischemic stress in murine cells and both murine and human kidney,3 residues were phosphorylated, whereas the other 2 sites werede-phosphorylated (FIG. 2D). Several of the site-specific andcompartment-specific phosphorylation changes occurred within or adjacentto NPM functional domains (FIG. 4). Two of 5 phospho-events (S88 andT95) detected in ischemic PTEC have been reported to cause cell death byregulating NPM localization and de-oligomerization in AML patients thatdetermine prognosis 21,69. However, the function of the other 3 NPMphospho-sites altered by stress (T86, T232 and S240) is unknown. Evenmore surprising, mass spec revealed identical phospho-events and NPMconsensus sequences at the same 5 residues in primary murine and humanPTEC after ischemic stress as detected in ischemic murine and humankidneys (FIG. 2D). Specifically, NPM phosphorylation in the healthykidney was identical to healthy PTEC and to non-ischemic murine kidney(data not shown). In contrast, the ischemic human kidneys exhibitedidentical phospho-changes that replicated phospho-events in ATP depletedPTEC and ischemic murine kidney (with the single exception of T86; FIG.2D). As a result of these exciting new findings in human kidneys it ishighly likely that therapeutics proven in our animal model can translateto effective AKI clinical trials.

Based on this phospho-proteomic data, 32 site-specific, amino terminal,flag-tagged NPM proteins with differential phosphorylation weregenerated (see FIG. 4B) and placed in a lentiviral vector (Baxphospho-mimic proteins; ref Wang, Havasi et al. 2011). Both the plasmidsequence and protein expression were confirmed. Serine or threonineresidues in each of the NPM phospho-mimics was rendered eithernonphosphorylatable (alanine substitution) or phospho-mimetic (glutamicacid substitution) at each of the 5 NPM residues identified by mass specto be altered by ischemia. These NPM phospho-mimics represent all of thepossible combinations of constitutively non-phosphorylated andphosphorylated variants at all of the 5 phosphosites (25 total mimics,see table, FIG. 4B). The flag epitope facilitates immunohistochemistryfor detecting NPM distribution, NPM oligomerization andimmunoprecipitation (IP) for detecting interaction between Bax and eachNPM phospho-mimic. To establish a causal link between phosphorylationand NPM-function, a flag-tagged NPM mimic that replicated the NPMphospho-state in healthy PTEC and kidney tissue (#10, “normal NPMmimic”) or replicated NPM phosphorylation in post-ischemic cells andtissue (#11; “stress-NPM mimic”; FIG. 4B) was introduced into primarymurine PTEC for further study. Dramatic differences in the biologicbehavior of these 2 NPM phospho-mimics were detected. Specifically, thestress NPM mimic exclusively localized to the cytosol (FIG. 5A) andcaused marked NPM de-oligomerization (FIG. 5B) in the absence ofischemic stress. During ATP depletion, the stress NPM mimic markedlyincreased NPM-Bax complex formation (FIG. 5C), dramatically increasedNPM accumulation in isolated mitochondria (FIG. 5D) and significantlydecreased PTEC survival after stress (FIG. 5E; n=4; P<0.05). Incontrast, the normal NPM-phospho-mimic exclusively localized to nucleoli(FIG. 5A), remained in the oligomerized state (5B), minimally complexedwith Bax during stress (FIG. 5C), and did not accumulate in isolatedmitochondria (FIG. 5D). In contrast to the stress NPM phospho-mimic, thenormal mimic actually improved PTEC survival, suggesting that itinterfered with cell death caused by native NPM. Using this phospho-datagenerate 2 new therapeutic peptides that target either T86, S88 and T95or T232 and S240 were generated (FIG. 4A) and tested (FIG. 7). Screeningthe remaining NPM mimic proteins can identify the specific function ofall 5 phospho-sites. These results demonstrate that synthesis ofpeptides that interfere with a single phospho-site are usefultherapeutics and provide a platform for testing the efficiency of NPMinhibitory peptides and peptide cocktails.

Determine the functional role of stress-induced NPM phosphorylationevents and renal cell death during ischemic AKI. First, the function ofeach of the 5 NPM serine/tyrosine phospho-sites identified in human andmurine cells subjected to ischemic stress (ATP depletion or hypoxia) areassessed. To streamline screening, the effect of each of the NPMphospho-mimic protein on PTEC survival after ATP depletion or hypoxia isdetermined (FIG. 1). The 3 most protective and toxic NPM phospho-mimicsare subjected to a panel of 5 bioassays of NPM function (described inFIG. 5A-5E). A chromatin binding nuclear release assay of the nuclearcell fraction68 can be used to identify the phospho-change(s) thatmediate NPM release from nucleolar DNA/histones, the first step in thecell death pathway. Since human primary PTEC are limited in supply andinformation in murine PTEC can guide in vivo experiment in mice, bothprimary cell lines are tested. To quantify compartment-specific changesin NPM phosphorylation caused by ischemic stress in vitro and in vivo,the phosphorylation mass at each phospho-site is measured in NPMharvested from primary human and murine PTEC subjected to ATP depletionor hypoxia using Stable Isotope Labeling by Amino acids in Cell culture(SILAC) as previously described. Mass tolerance can set to 0.1 Da for MSand MS/MS, respectively and a threshold for significant threshold can beset at P<0.05 to distinguish between correct and false peptideidentifications in 3 separate samples from individual experiments. Cellcompartment-specific analyses for the appropriate peptide size shift forsite-specific serine/threonine phosphorylation can be analyzed. Anunderstanding of the magnitude and cell compartment specificity of eachNPM phospho-change was used to characterize the NPM-Bax cell deathpathway and inform the development of specific peptides directed at oneor more differentially phosphorylated NPM sites that regulate NPM-Baxtoxicity.

Identify NPM interacting proteins as therapeutic targets in AKI. Toidentify NPM binding partners (including Bax, FIGS. 9A-9B) and optimizethe treatment and prevention of AKI, mass spectrometry forprotein-protein interaction can be performed from resting human andmurine and PTEC lysates using an NPM antibody (Abeam; Cambridge, Mass.)before and after ATP depletion or hypoxia. Specifically, proteins thatinteract with the 3 most toxic NPM mimic proteins and the 3 mostprotective NPM mimics were subjected to mass spec performed underconditions that preserve protein-protein interactions, an approachsuccessfully used to detect protein interactions in intact cells. Thisapproach identifies both protective and toxic NPM interacting proteins.Since NPM-Bax interaction occurs during ischemic stress, analysis of theNPM immunoprecipitates is performed to ensure detection of Bax (as apositive control) before submitting samples for mass spectrometry toidentify other NPM binding partners. The stringency of the conditionsused to IP NPM from PTEC can be adjusted until Bax is detected in NPMimmunoprecipitates to increase the yield of other NPM binding partnersthat represent new therapeutic targets for interfering with the NPM celldeath pathway during ischemic stress. Recently, Hsp90 and Hsp70,stress-induced proteins that regulate cell survival, have beenphysically linked to NPM, and are likely to regulate NPM-Bax toxicity.In addition, both BAG-1 (an Hsp70 binding protein) and CRM-176 have beenreported to interact with, and regulate NPM. Therapeutics directed atNPM interacting proteins can also be selected as NPM inhibiting agents,as described herein that can ameliorate NPM-Bax complex formation andtoxicity in a manner similar to the disclosed therapeutic NPM inhibitorpeptides (FIG. 7).

Herein, the inventors have demonstrated the following (1) a direct linkbetween site-specific NPM phosphorylation events of NPM polypeptide andits NPM contribution to cell death; (2) demonstrated location ofquantitative phospho-site amino acids that can be blocked or interferedwith for the development of NPM inhibitor peptide therapeutics; and (3)identified NPM binding partners during ischemia to identify additionaltherapeutic targets for testing in vitro and in vivo. Given the clearresults provided by the normal and stress mimic NPM phospho-mimics ineach of the 5 bioassays, the inventors tested 30 remaining NPMphospho-mimics (FIG. 4B) in murine and human PTEC to identify thespecific sites(s) that regulate each NPM function (data not shown).Although a single phospho-event regulates both NPM de-oligomerizationand nuclear NPM translocation in AML patients, it is likely that acombination of stress-induced phosphorylation events regulate some NPMfunctions, and therefore the inventors assessed all potentialphosphocombinations (FIG. 4B). The inventors also envision blocking theinteraction of NPM interacting proteins to inhibit NPM-Bax formation, asNPM-Bax undergoes mitochondrial translocation and NPM also lacks amitochondrial localizing sequence. Finally, identification of NPMinteracting proteins that bind to protective NPM mimics protein can beused to cytoprotection. For example, if interaction between a toxic NPMmimic and a specific protein is detected by mass spec, then one can usean established algorithm to identify its NPM interaction domain and awebsite is available (http://cmbi.bjmu.edu.cn/huphospho). Onceidentified, a NPM inhibiting or blocking peptide can be synthesized andtested.

While ischemic stress in vitro does not replicate ischemia in vivo,herein, the inventors now demonstrate show that virtually identicalphospho-events occur after ischemic stress in mice and human PTEC as inischemic murine and human kidneys (FIG. 2D), cells are appropriate fortesting the efficacy of our therapeutic agents and to inform animalstudies. Of the 65 potential post-translational modifications detectableby mass spectrometry, the inventors discovered that serine/threoninephosphorylation regulates NPM toxicity during cell stress. As aninexpensive and rapid alternative to mass spec, commercially availablephospho-antibodies can be used to detect site-specific NPMphosphorylation. Although an anti-T95 phospho-antibody is presentlyavailable (AbCam, Cambridge, Mass.), one can of ordinary skill in theart can readily generate new NPM phospho-specific antibodies directedagainst T86, S88, T232 and S240 using a commercial vendor (for example,see:“thermofisher.com/us/en/home/lifescience/antibodies/custom-antibodies/custom-antibody-production/custom-monospecific-antibodyproduction”).Such phospho-specific Abs can be used to distinguish toxic fromnon-toxic forms of NPM in the urine and other biological samples fromanimals and patients, e.g., human subjects after renal ischemia.

To What Extent does Antagonizing NPM-Bax Prevent Renal Epithelial CellDeath and Tissue Injury?

The inventors have determined that ischemic stress converts NPM from acellular friend to a cytotoxic foe that collaborates in lethal “BaxAttack” (FIG. 10). In human disease, it is unusual for singleintervention to achieve therapeutic success. However, strong preliminarydata show that interventions directed against NPM-Bax significantlydecrease injury in primary human PTEC and participate in ischemic injuryin human kidneys. However, a combination of agents that antagonizeNPM-Bax will be more effective than if each is used alone. Afteridentifying the NPM post-translational phosphorylation eventsresponsible for NPM-Bax toxicity and NPM interacting proteins,site-specific therapeutic peptides (e.g., Peptides #1, #2 and #3disclosed herein) can be combined with pharmacologic agents, can be usedto disrupt NPM-Bax toxicity and to optimize human and murine PTECsurvival as well as the viability of human and murine kidney tissuedelivered either before or after ischemic stress. Exemplary compounds oragents to be combined are shown in FIG. 11. The inventors selected thereagents to be combined based on performing an in vivo study with Baxblocking peptide and GGA, reagents with distinct mechanisms of action(data not shown). A Peptide #1 and GAA combination was significantlymore reno-protective than either agent alone in mice subjected to renalischemia (data not shown).

NPM is a relevant therapeutic target in human AKI. Primary humanproximal tubule epithelial cells (PTEC) derived from cadaveric kidneyswere subjected to ischemic stress (transient ATP depletion) in thepresence of specific NPM siRNA or non-sense siRNA. This maneuver reducedsteady state NPM content by 50% in PTEC lysates and significantlyincreased PTEC survival after ischemic stress (FIG. 6; P<0.05; n=6).Thus, manipulating NPM is an effective maneuver. Using mass spec data,the inventors generated 2 new, water soluble peptides that penetraterenal cell membranes (SEQ ID NO: 8-10) and significantly improve PTECsurvival after ischemic stress (synthesized by Biomatik, Cambridge,Ontario). Small peptides of 30 amino acids or less are sufficientlyspecific to minimize off target effects and also reduce the risk ofeliciting an antibody response that causes tachyphylaxis. Peptides werecommercially synthesized and fused to a nuclear localizing sequence(this NLS has no functional relationship to the “NPM NLS” that regulatesNPM translocation). The NLS-CPP markedly increases renal accumulation ofsmall peptides8 as well as in the proximal tubule. Compared to control(a random amino acid sequence fused to the NLS and CPP), the Baxblocking peptide (#1) significantly improved cell survival by 25%(P<0.05; n=5). To determine its effect on NPM-Bax complex formation,active Bax was immunoprecipitated from PTEC rendered ischemic with anantibody directed against the 6A7 epitope. The precipitates weresubjected to immunoblot analysis using an anti-NPM antibody. Compared tocontrol (a size matched random peptide), the Bax blocking peptide(Peptide #1) decreased NPM-Bax complex formation by about 50% inischemic primary murine PTEC as well as in renal cortical homogenateharvested from a murine kidney after ischemia caused by pedicle clamping(FIG. 9A-9B). NPM and Bax accumulation was measured in mitochondriaisolated from human PTEC after ischemic stress. The Bax blocking peptidedecreased mitochondrial Bax accumulation to a greater extent than itreduced NPM accumulation, especially after ischemic stress P<0.05, n=4;upper panel). This data strongly demonstrates that NPM-Bax complexformation as a prerequisite for Bax-mediated outer membrane injury thatcauses PTEC death1. Taken together these results show that NPM-Baxcomplex formation accompanies ischemic stress in vitro and in vivo andthat our Bax blocking peptide partially decreases complex formation andreduces mitochondrial Bax accumulation. Thus, the inventors havedemonstrataed that increasing the peptide dose above 2 mg/animal (7 μM)or administering a second dose within 24 hr did not provide additionalprotection in ischemic PTEC or mice. This is likely due to the prolongedhalf-life (about 80 hr) of the peptide in vitro and in vivo due to thepresence of an AC modification slows degradation.

To generate reagents for testing as a therapeutic cocktail, new peptidesthat either replicate all 3 amino-terminal NPM phosphorylation sites(T86, S88 and T95; FIG. 4A) or both carboxy-terminal phosphorylationsites (T232 and S240) that are altered by renal ischemia in vivo (FIG.2D) were also tested in vitro. Both peptides significantly improvedhuman PTEC survival after ischemic stress (P<0.05, n=4). Thus,renal-targeted peptides that potentially block multiple NPMphospho-sites during renal ischemia are an effective approach forameliorating NPM-Bax toxicity and improving PTEC survival. Thisdemonstrates additional NPM phospho-proteomics and additional peptidescan be developed that interfere with NPM-Bax toxicity and ameliorateischemic AKI in humans.

The inventors have also tested pharmacologic agents that disrupt NPM-Baxat specific steps in the cell death pathway (outlined in FIG. 11). Theseagents include geronylgeronylacetone (GGA), a potent Hsp70 inducer86,avrainvillamide, and NSC34888437. Importantly, exposure to the IC50 doseof these agents does not alter PTEC morphology or cause significanttoxicity after 48-72 hr (data not shown). The inventors recentlyreported that GGA induces Hsp70 in murine PTEC and in the intact murinekidney. Hsp70 markedly reduces NPM-Bax complex formation during ischemicstress by 2 distinct mechanisms: a nucleolar-restricted Hsp70 mutantlimits NPM translocation into the cytosol, whereas a cytosol restrictedHsp70 mutant sequesters cytosolic (data not shown).

Importantly, GGA-mediated cytoprotection requires Hsp70, sinceprotection is not observed in PTEC harvested from Hsp70 knockout mice,confirming that Hsp70 itself protects against NPM-Bax toxicity. The roleof Hsp70 (and Bag-1, an Hsp70 regulatory protein) as a potential NPMinteracting protein is assessed by the inventors and the inventorsdiscovered that GGA augments reno-protection afforded by the Baxblocking.

Historically, therapy derived from cell culture or animal models hasfailed to translate to human AKI. To address this challenge, theinventors provide data in human cells and tissue. The inventors comparedthe degree of NPM translocation from the nuclear to the cytosolic cellfractions of primary murine and human PTEC subjected to ischemic stress(ATP depletion or hypoxia, an alternative ischemia model). In bothcases, ischemic stress markedly increased cytosolic NPM content inmurine and human primary PTEC. A marked increase in cytosolic NPM wasalso detected in renal cortical homogenates obtained from ischemiccadaveric kidneys rejected for transplantation form two donors. Onekidney form each donor has perfusion failure, was grossly ischemic andexhibited severe proximal tubular injury. The other kidney in each pairwas well perfused without ischemic injury. Cytosolic NPM accumulationwas markedly increased in cortical homogenates harvested from theischemic human kidneys vs. control (FIG. 3B-3C). Therefore, theinventors demonstrate that NPM leakage, the first step in the NPM-Baxcell death pathway occurs after ischemic stress in vitro and in theintact human kidney.

Combining peptides and pharmaceuticals to maximally reduce NPM-Baxtoxicity in vitro. To streamline the experiments, single therapeuticpeptides and pharmaceuticals with distinct mechanisms of action werefirst be screened for their pro-survival effect in murine PTEC subjectedto ischemic stress (ATP depletion or hypoxia) using a high throughputMMT assay in 96 well plates. Based on these studies, a cocktail of themost effective peptides with distinct mechanisms of action were tested(e.g., the Bax blocking peptide (peptide #1) that interferes withNPM-Bax complex formation are combined with peptides (peptides #2 and#3) that interfere with toxic NPM phospho-events. The most effectivepeptide cocktail are be combined with pharmaceuticals that either reduceNPM translocation (avaranillamide and GGA), de-oligomerization (NSC34884and an specific RNA aptamer described by others) (see FIG. 11), andNPM-Bax complex formation (Bax and NPM peptides), and mitochondrialNPM-Bax translocation (peptide(s)) to improve PTEC survival afterischemic stress is assessed. The most effective therapeutic combinationsare tested for their pro-survival effect in ischemic primary human PTEC.The effect of these therapeutic maneuvers on NPM-Bax toxicity isassessed using bioassays of NPM function and correlated with renal celldeath caused by apoptosis and necrosis. Apoptosis is detected usingassays of outer mitochondrial membrane injury (apoptosis inducing factor(AIF) and cytochrome c leakage), caspase 3 activity, and morphologicevidence of apoptosis in cells stained with Hoechst, the “gold standard”for detecting condensed chromatin and apoptotic bodies. Necrosis canalso be quantified using propidium dye uptake and LDH release (markersof cell membrane injury). To more rapidly assess the type of cell deathafter ischemic stress, the inventors adopted a new screening technique,where the assay uses the Celigotm image cytometer to rapidly distinguishapoptotic from necrotic cells stained with propidium iodide, Hoechst dye#33342 and annexin V. The inventors demonstrate that Celigo measures ofcell death (apoptosis+necrosis) replicate the MTT assay (data notshown). To assess their efficacy for treating ischemic stress, theexposure to the most effective combination(s) for preventing ischemicPTEC injury in vitro, is progressively delayed after ischemic stress.This approach inform in vivo treatment of AKI, and provides insight intothe therapeutic window for AKI treatment and reduce the number of animalexperiments. Effective therapeutic Interventions derived from cellsculture experiments is selectively tested in fresh, 100 micron thickhuman and murine kidneys slices subjected to ischemic stress. Thisexperimental model has been extensively used to simulate ischemia invivo and has the benefit of including multiple renal cell types andintact kidney architecture95,96. To be suitable for testing, tissueslices will require: minimal or no evidence of acute injury (minimaltrypan blue staining and absent or minimal cytosolic NPM accumulation).Tissue slices will be subjected to hypoxia or exposed to metabolicinhibitors that reduce cell energy content. Measures of cell death(trypan blue, Hoechst, and annexin V staining as well as LDH release)and histologic injury score will be correlated with the effect oftherapeutics on each of the NPM functional bioassays. The impact of themost effective therapeutics on NPM phosphorylation will be assessed bymass spec. To identify their target of protection in murine and humankidney tissue, fluorescent-labeled peptides (synthesized by Biomatik)will be used to assess their intra-renal distribution in 10 microntissue sections.

Generation of additional peptide therapeutics for antagonizing NPM-Bax.Since the Bax blocking peptide (peptide #1) reduces but does noteliminate NPM-Bax interaction, the inventors generated a new peptidethat competitively inhibits the NPM binding site for Bax (described inExample 1). To selectively inhibit each regulatory NPM phospho-site,fluorescent labeled peptides that replicate their consensus sequencesand the regions flanking it are designed and tested as reported forother peptides. The phospho-site (initially 10-20 amino acids in lengthflanking the 5-10 amino acids adjacent to each of the 5 identifiedphospho-sites) are produced. The competitive NPM inhibitor peptide ofinterest can optionally be fused to the NLS sequence and/or a cellpenetrating peptide (“CPP”) that markedly enhances renal uptake of ourcompetitive Bax peptide mimotope, creating a therapeutic peptide of20-30 residues in length. Since the ischemic phospho-NPM mimic stronglyinteracts with Bax, it can be used to identify the specific phospho-sitethat regulates NPM-Bax interaction. A peptide directed against NPM willalso disrupt NPM-Bax interaction, further decreasing NPM-Bax interactionand cell toxicity.

Identification of the mechanism of action of peptides andpharmaceuticals directed against NPM-Bax. The mechanism of action foreach of our interventions on NPM-Bax can be assessed by complimentaryapproaches including mass spec to identify their impact on post-ischemicNPM phosphorylation events, NPM phosphospecific antibodies, and NPMfunctional bioassays. A mammalian 2-hybrid system combined with aLuciferase Reporter Assay System (CheckMate™ and Dual-LuciferaseReporter Assay System; Promega, Madison, Wis.) was used to assessinteraction between the most toxic and protective NPM mimic with Baxmammalian cells and kidney tissue. This assay was used in cells andkidney slices to demonstrate the degree to which peptides andpharmaceuticals disrupt the NPM-Bax complex and characterize theirmechanism of action. For example, the therapeutic peptides #2 and #3could competitively inhibit interaction between NPM and the kinases orphosphatases that regulate NPM phosphorylation events during ischemia.Alternatively, these peptides could act as decoys for the regulatorykinase or phosphatase, without requiring them to interact with NPM. Tomonitor stress-induced NPM translocation in live cells and the effect ofinterventions to inhibit it, PTEC are infected with lentivirus thatcontains wild type and mimic NPM fused to DS red (Addgene, Cambridge,Mass.) at the carboxyNPM terminus. Flag or DS red tagged NPM mimics willreplicate baseline vs. stress-induced changes in phosphorylation at T86,S88, T95, T232, and/or S240 singly and in all possible combinations(FIG. 4A-4B).

The inventors have developed peptides that block NPM-proteininteractions regulated by NPM phosphorylation events. One can combinereagents that act on distinct steps in the NPM-Bax pathway (FIG. 11) tooptimally antagonize NPM-Bax. Specifically, PTEC survival, tissueviability and histologic score can be improved by simultaneouslyreducing NPM translocation, de-oligomerization, NPMBax complexformation, and mitochondrial accumulation of the NPM-Bax complex. Onecan first identify the most effective reagent (peptide orpharmaceutical) that antagonizes each toxic step and then to combinethem. NPM bioassays are correlated with PTEC survival in vitro andhistologic injury in tissue slices in the most effective combinations.The mammalian 2-hybrid system can quantify the protective effect ofcombined peptide and pharmaceuticals for preventing and treating AKI.Although existing evidence is stronger that NPM-Bax mediates apoptosis,recent evidence shows that necrosis converges at the outer mitochondrialmembrane, involve Bax, and that the latter form of cell death isregulated. Therefore, antagonism of the NPM-Bax cell death pathway willdecrease both apoptosis and necrosis.

Cell death pathways other than apoptosis and necrosis exist. However,minimal evidence suggests that these pathways contribute to ischemic AKIin humans. In addition, these pathways lack a distinctive morphology(unlike apoptosis or necrosis) and at present, are either biochemicallydefined (e.g., by RIP3 kinase activation) or are implicated by theirresponse to inhibitors (e.g., necrostatin). However, phosphorylationregulates some of these pathways and would be amenable to proteomicanalysis by mass spec to inform the development of interfering peptidesfor this alternative cell death pathway. As discussed herein, proteomicsis used to reveal the actual NPM binding site for Bax, to develop apeptide antagonist. In addition to use of mass spectrometry to revealthe phospho-site that regulates Bax binding, serial NPM truncation (NPMis a 292 aa protein) can be performed to evaluate the region on NPM thatbinds to Bax.

Example 6

Evaluate NPM Phosphorylation in the Diagnosis, Treatment and Preventionof AKI In Vivo.

Preliminary data show that NPM-Bax is a highly promising target in humanAKI. Data gathered in Examples 1 and 2 can be used to improvetherapeutic efficacy beyond that afforded by the Bax blocking peptide(Peptide #1) alone, extend the time window for treating AKI (currently 3hr) and assess the impact of therapeutics on apoptosis and necrosis,forms of death detected in human AKI. Unfortunately, renal function(BUN/Cr) are imperfect for rapidly or accurately diagnosing AKI and GFRloss fails to detect nearly 45% of human AKI. Urinary biomarkers haveimproved AKI diagnostics but have not been clinically useful in theabsence of effective human therapy. To address this concern, the KidneyResearch National Dialogue sponsored by the NIDDK suggested newbiomarkers and tissue assays that can be translated to human AKI and arelinked to its pathogenesis would be useful.

In sum, an ideal AKI biomarker described herein: (1) rapidly andaccurately detects AKI; before a change in GFR or urine output; (2)reflects AKI severity (need for renal replacement therapy and thelikelihood of recovery); (3) triggers therapy; (4) guides optimal dosingof reno-protective agents. To address these challenges, the inventorshave combined two newly developed in urine and in human kidneys biopsytissue for detecting NPM. Combined with assays of differential NPMphosphorylation this novel biomarker is useful for detecting ischemic aswell as nephrotoxic AKI, common diseases (aminoglycosides, vancomycin,cisplatin and radiocontrast agents) in which Bax (and therefore NPM)have been reported to play a pathogenic role.

The renal-targeted Bax blocking peptide (peptide #1) reduces AKIseverity by 40-50% in mice when administered before the insult. Todetermine its efficacy for treating ischemic AKI, the inventors delayedIV peptide administration for 0-6 hr after releasing the pedicle clamp.Compared to control (random peptide), the Bax blocking peptide,significantly improved BUN by 15-35% on days 1-6 if given within 3 hrafter 22 min bilateral renal ischemia. Although 30% of control mice diedof severe AKI, 98% of peptide-treated animals survived. As intended, theBax blocking peptide decreased NPM-Bax interaction in renal corticalhomogenates by about 50%, an effect similar to the degree ofreno-protection. This data demonstrates that more effective therapydirected at the NPM-Bax pathway is both rational and feasible.

To test the hypothesis that therapeutics with distinct mechanisms ofaction on NPM-Bax improve AKI outcome, the inventors combined a singledose of our Bax blocking peptide with GGA, a non-toxic agent widely usedin Asia for dyspepsia that also induces Hsp70 and is reno-protective.This is an ideal drug combination since the Bax peptide reduces NPM-Baxcomplex formation (data not shown) and GGA reduces cytosolic NPMaccumulation during ischemic stress exclusively by inducing Hsp70 (datanot shown). After 28 min of bilateral pedicle clamping, a severe renalinsult, Bax peptide plus GGA was significantly more protective thaneither agent alone on renal function measured on days 1-7 (data notshown). Remarkably, control mice had a mean BUN level of 140 mg/dl andall animals died within 48 hr after ischemia. In contrast, mice treatedwith Bax peptide, GGA or the combination of these agents had a mean peakBUN of only 100m/dl and all survived! Thus, therapy that antagonizesNPM-Bax at multiple steps is more effective in protecting renal functionthan a single agent and improves survival, the rodent endpointequivalent to a patient requiring dialysis.

To improve AKI diagnostics and assessment of its severity, the inventorsherein have developed a urine assay to detect NPM released by ischemicrenal cells. In brief, urine supernatant was assayed for NPM afterintact cells were removed by differential centrifugation (1,500 RPM×5min). In normal mice at baseline as well as sham operated mice (n=3),urinary NPM is virtually undetectable. In contrast, urinary NPM isabundant after 22 min bilateral pedicle clamping detected by a specificNPM antibody (Sigma-Aldrich, St. Louis, Mo.) using a semi-quantitativedot blot assay (FIG. 3A; n=9). In fact, NPM appeared within 12 hr andpersisted for at least 24 hr after ischemia. NPM was also detected inthe tubular lumen of an ischemic human kidney (FIG. 3B-3C). Remarkably,4 of 9 ischemic mice with the highest urinary NPM content died within 48hr. These data show for the first time that urinary NPM may be: (1) aneffective biomarker for proximal tubule cell death; (2) predict theseverity of organ injury and (3) be a suitable assay for optimizing thedose of our therapeutics in murine and human AKI. Quantitative urinarydot blot testing with phospho-specific NPM antibodies that exploitdifferential phosphorylation (as detected in normal and ischemic murineand human kidneys; (FIG. 3A) may be an even more accurate marker of AKIonset, severity and recovery.

To assess the feasibility of using NPM as a diagnostic marker in humanAKI, NPM immunohistochemistry (IHC) and Hoechst staining was performedon fresh frozen 10 micron thick human kidney tissue sections obtained byroutine biopsy. Blinded specimens from 3 patients were examined withnormal appearing tubules or mild acute tubular injury or severe acutetubular injury. In this study, NPM was exclusively localized to thenuclei of healthy proximal tubule cells (FIG. 3C). In contrast, NPM wasdiffusely distributed in cytosol or lumen of damaged tubular cells; thenuclear region was devoid of NPM (FIG. 3C). Interestingly, the degree ofcytosolic NPM staining positively correlated with tubular injuryseverity (data not shown), whereas NPM in medullary tubules remainednuclear. Remarkable, NPM redistribution occurred before Hoechst stainingrevealed evidence of either apoptosis or necrosis. Thus NPMtranslocation may be an early diagnostic marker that ispathophysiologically linked to renal death and organ failure.

TO what extent do maneuvers that antagonize NPM and Bax ameliorateischemic AKI? The prevention and treatment of in vivo renal ischemia canbe based on in vitro studies of the single most effective peptide andpharmaceuticals that individually decrease each step in the NPM-Bax celldeath pathway (FIG. 10) as assessed by our NPM bioassays. The reagentdose and timing is guided by in vitro experimental results, publishedIC50 data for GGA, the RNA aptamer dose successfully used in mice andthe inventors prior experience with the Bax peptide in murine AKI.Renoprotection afforded by single or combined therapeutic is assessed byserial serum BUN/Cr measured for 7 days post-ischemia, a time frameadequate to encompass the peak severity of AKI and death or recovery.For maneuvers that afford significant reno-protection, studies wererepeated and mice sacrificed at 24 hr post-ischemia to perform acomprehensive, blinded histologic injury score (including an estimate ofleukocyte infiltration) as previously described. The most effectivemaneuvers that prevent ischemic AKI are further tested as potential AKItreatment. To establish the therapeutic window for treating AKI,combined therapy is progressively delayed for 1-12 hours post ischemiaor until significant reno-protection is no longer observed. To assessthe degree to which of the NPM inhibitor agents/therapeutics interferewith steps in the NPM-Bax cell death pathway, NPM bioassays described(FIG. 5A-5E plus a DNA/histone binding release assay are repeated inrenal cortical homogenates (as described above). Renal uptake and theintra-renal distribution of peptides that disrupt NPM-Bax interaction isassessed using fluorescent-tagged Bax and NPM peptides (synthesized byBiomatik). Apoptosis and necrosis were quantified using multiplemethods. First, blinded fresh kidney tissue sections are stained withHoechst 33342 and PI, a vital dye, followed by fixation and TUNELstaining as described in the ischemic kidney in vivo122.

Second, pan-caspase activity, a direct measure of active apoptosis infixed tissue is measured with a kit (CAS-MAP by Vergent Bioscience,Minneapolis, Minn.). Since necrosis and apoptosis likely vary with theischemia severity, renal pedicles are clamped for 25, 30 or 35 min toassess their relative contributions to progressive organ failure andmortality. After injection of paraformaldehyde in situ, the distributionof the fluorescent label is assessed by routine histochemistry in 10micron thick frozen kidney tissue sections as described by us fora2-microglobulin. These studies identify interventions that disrupt NPMtoxicity, inhibit apoptosis and necrosis, characterize their mechanismof action, identify the intra-renal structures protected by NPM and Baxpeptides, and optimize therapeutics for treating and preventing ischemicAKI in a model that replicates the NPM-Bax toxicity in human AKI.

Using NPM and differential NPM phosphorylation in a urine and tissue asa diagnostic and prognostic assay for AKI. Urine collected from control,sham-operated and post-ischemic mice are accessed by simple bladdercompression and analyzed for NPM content using quantitative dot blotanalysis, a technique that measures total urinary NPM independent ofurine volume. Total urine NPM can be correlated with kidney function(BUN/Cr), histologic injury score and animal survival. The time windowfor detecting urinary NPM is tested between 0-48 hr after ischemia andis compared with established urinary AKI biomarkers including KIM-1, NAGand NGAL125-128 that in combination, predict AKI with greater accuracythan GFR markers. In addition to total urinary NPM content,site-specific phosphorylation NPM phosphorylation is measured inischemic human and murine kidneys and murine urine by quantitative dotblot using site-specific phospho-antibodies. This urinary NPMphospho-data is correlated with kidney tissue NPM bioassays, kidneyfunction, histology, and animal survival. To determine the extent towhich these new NPM assay predicts renal recovery, total and phospho-NPMis probed with site-specific NPM-phospho-antibodies in the murine urineafter the administration of effective therapeutics to detect each toxicphosphorylation event (FIG. 2D). This study demonstrates that NPM andphospho-NPM with ischemic AKI severity and facilitate the use ofnon-invasive (urine based) assays to substitute for invasive (tissuebased) testing in ischemic AKI.

To identify causes of human AKI amenable to NPM-Bax therapy, NPMredistribution into the cytosol, the first step in NPM cell deathpathway, is assessed by IHC in human kidney biopsy specimens obtainedfrom patients with ischemic or nephrotoxic AKI of diverse etiologies andcompared to controls without tubular injury. Blinded tissue samples aresubjected to routine IHC with semi-quantitative histologic scoring oftubular injury. Tissue samples are probed with an NPM antibody to assessNPM localization and with site-specific NPM phospho-antibodies to assessthe intra-renal distribution of NPM with toxic phosphorylation eventsthat should replicate the toxic vs. normal NPM mimic; FIG. 5A). At least10 random fields in each specimen with be analyzed with an automatedscanning microscope (BU core imaging facility) using averaged thresholdmeasure (ATM) to objectively score tissue staining. NPM redistributionin the proximal, distal and collecting ducts are compared in renalcortex, juxtamedullary region and the medulla of ischemic mice as wellas in blood vessels and endothelium to assess the effect of ischemia onrenal NPM.

Given that the inventors have demonstrated that combined therapy issuperior to single agent, one easily can use a strategy of a combinationof agents, e.g., as shown in FIG. 11 with one or more of the NPMinhibitor agents (e.g., including but not limited to inhibitor peptidescomprising SEQ ID NO: 1-3 or peptides having 85% sequence identitythereto) to increase reno-protection for preventing ischemic AKI and toextend the time window for its treatment as described for ischemicstroke or acute myocardial ischemia. Reducing NPM-Bax toxicity decreasesboth apoptosis and necrosis in vivo and that in combination, moreaccurately predicts GFR than necrosis alone. Also, urinary NPM increasesthe sensitivity of existing urinary AKI biomarkers. This assay detects afall in total and phospho-urinary NPM that is likely to reflect effecttherapy directed against NPM-Bax in mice and humans. Finally, NPMtranslocation is detected in forms of nephrotoxic AKI in which Bax hasbeen implicated to cause tubular injury before GFR or urine outputdeteriorate, triggering early, effective intervention with therapeuticsto ameliorate NPM-Bax toxicity. Since post-translational NPMmodification uniformly detects ischemic renal injury in PTEC and kidneytissue (FIG. 2D), use of phospho-NPM is a more sensitive test forNPM-toxicity than total NPM. Since urinary NPM persists in ischemic micefor at least 24 hr (FIG. 3A), quantitative urinary total and phospho-NPMallows one to establish a threshold values to accurately predict theseverity and reversibility of ischemic AKI in mice (and ultimately inhumans). Since NPM distribution strikingly differs in normal vs. damagedproximal tubules (and medullary tubules), NPM also identifies thespectrum of AKI patients in whom NPM-Bax therapeutics are likely to beeffective. The inventors described urine and tissue assays that providemechanistic insight into AKI pathogenesis, and allow the subject to beadministered the NPM peptide/pharmaceutical therapy as described hereinand also may be useful to predict renal recovery.

Although BUN/Cr do not accurately reflect fluctuating GFR113, othershave used these parameters to approximate peak GFR loss for timinghistologic and biochemical analyses. One will be able to detect at leastsome site-specific NPM phospho-changes using phospho-antibodies.Alternatively, one of ordinary skill in the art can perform mass spec ofurinary NPM as described herein (see FIG. 2A-C). Ameliorating NPM-Baxtoxicity will reduce inflammation detected in kidney tissue. One canalso perform further analysis of resident dendritic cell activation,infiltrating neutrophils and macrophages, CD4+T and B cells as well askiller T cells implicated in AKI using a flow cytometry assay toidentify inflammatory cells in minced fresh kidney tissue. Also, If NPMtranslocation is detected in kidney biopsies of patients withnephrotoxic AKI, one can assess other measures of NPM-Bax toxicityincluding Bax activation using a 6A7 specific Ab, and NPM-Bax complexformation (FIG. 10) in stored kidney biopsy tissue. Finally, since cellculture and rodent models of nephrotoxic AKI are readily available, onecan also directly assess our NPM-Bax therapeutics for preventing andtreating in nephrotoxic AKI in which NPM translocation occurs.

REFERENCES

All references cited herein and throughout the specification andExamples are incorporated herein in their entirety by reference.

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REFERENCES FOR EXAMPLE 5

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1. A kit comprising a pharmaceutically acceptable carrier and a peptide,wherein the peptide is selected from at least one of: a. (SEQ ID NO: 2)TLKMSVQPTVSLGGFEITPPVVLRLK,

b. (SEQ ID NO: 3) ESFKKQEKTPKTPKGPSSVEDIKAK,

c. a peptide having 95% sequence identity to SEQ ID NO: 2, d. a peptidehaving 95% sequence identity to SEQ ID NO: 3, or e. a peptide of any of(i)-(iv) fused to a renal nuclear localization signal (NSL), andinstructions for administration of the peptide to the subject for thetreatment of a human subject with a kidney injury, ischemia or a subjectafter ischemic injury.
 2. The kit of claim 1, wherein the instructionsfor administration of the peptide to the subject for the treatment of ahuman subject with a kidney injury, ischemia or a subject after ischemicinjury indicate the peptide should be administered to the subject within48 hours of an ischemic event or ischemic injury.
 3. The kit of claim 1,wherein the kidney injury is selected from the group consisting of:injury to the proximal tubule of the kidney; acute kidney injury (AKI);chronic kidney disease (CKD); and early kidney injury which willprogress into chronic kidney disease (CKD).
 4. The kit of claim 1,wherein the at least one peptide is packaged in any of: a sealed foil, aunit dose container, a blister pack or strip packs.
 5. The kit of claim1, wherein the NPM inhibitor peptide is fused to a renal targetingnuclear localization sequence (NSL).
 6. The kit of claim 1, wherein thepeptide is formulated for oral administration.
 7. The kit of claim 1,wherein the peptide is formulated in any one of the dosage forms:tablets, pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion.
 8. The kit of claim 1, wherein thepharmaceutically acceptable carrier comprises an excipient.
 9. The kitof claim 8, wherein the excipient is selected from the group of: water,glycols, oils, alcohols, flavoring agents, preservatives, and coloringagents, starches, sugars, microcrystalline cellulose, kaolin, diluents,granulating agents, lubricants, binders, and disintegrating agents. 10.The kit of claim 1, wherein the peptide is packaged in a pressurizedaerosol contained together with a suitable propellant.
 11. The kit ofclaim 1, wherein the at least one peptide is formulated as a powderacceptable for intrabronchial administration.
 12. The kit of claim 1,wherein the at least one peptide is formulated in a sustained-releasepreparation.
 13. The kit of claim 12, wherein the at least one peptidein a sustained-release preparation is the peptide in a sustained-releasefilm, matricies or microcapsule selected from any of: polyestermatricies, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers and poly-D-(−)-3-hydroxybutyricacid.